[Detection rates and high concentration of herpesvirus (Orthoherpesviridae) DNA in autopsy materials from patients with COVID-19 fatal outcome].

INTRODUCTION: SARS-CoV-2 infection causes immune disorders that create conditions for the reactivation of human herpesviruses (HHVs). However, the estimates of the HHVs effect on the course and outcome of COVID-19 are ambiguous. Аim - to study the possible relationship between the HHV reactivation and the adverse outcome of COVID-19. MATERIALS AND METHODS: Postmortem samples from the brain, liver, spleen, lymph nodes and lungs were obtained from 59 patients treated at the Moscow Infectious Diseases Hospital No.1 in 2021-2023. The group 1 comprised 39 patients with fatal COVID-19; group 2 (comparison group) included 20 patients not infected with SARS-CoV-2 who died from various somatic diseases. HHV DNA and SARS-CoV-2 RNA were determined by PCR. RESULTS: HHV DNA was found in autopsy samples from all patients. In group 1, EBV was most often detected in lymph nodes (94%), HHV-6 in liver (68%), CMV in lymph nodes (18%), HSV in brain (16%), VZV in lung and spleen (3% each). The detection rates of HHVs in both groups was similar. Important differences were found in viral load. In patients with COVID-19, the number of samples containing more than 1,000 copies of HHV DNA per 100,000 cells was 52.4%, in the comparison group - 16.6% (p < 0.002). An association has been established between the reactivation of HSV and HHV-6 and the severity of lung damage. Reactivation of EBV correlated with increased levels of liver enzymes. CONCLUSION: Reactivation of HHVs in patients with fatal COVID-19 was associated with severe lung and liver damages, which indicates a link between HHV reactivation and COVID-19 deaths.

Therapeutic vaccines for herpesviruses.

Herpesviruses establish latent infections, and most reactivate frequently, resulting in symptoms and virus shedding in healthy individuals. In immunocompromised patients, reactivating virus can cause severe disease. Persistent EBV has been associated with several malignancies in both immunocompromised and nonimmunocompromised persons. Reactivation and shedding occur with most herpesviruses, despite potent virus-specific antibodies and T cell immunity as measured in the blood. The licensure of therapeutic vaccines to reduce zoster indicates that effective therapeutic vaccines for other herpesviruses should be feasible. However, varicella-zoster virus is different from other human herpesviruses in that it is generally only shed during varicella and zoster. Unlike prophylactic vaccines, in which the correlate of immunity is antibody function, T cell immunity is the correlate of immunity for the only effective therapeutic herpesvirus vaccine-zoster vaccine. While most studies of therapeutic vaccines have measured immunity in the blood, cellular immunity at the site of reactivation is likely critical for an effective therapeutic vaccine for certain viruses. This Review summarizes the status of therapeutic vaccines for herpes simplex virus, cytomegalovirus, and Epstein-Barr virus and proposes approaches for future development.

First detection of Koi herpesvirus disease (KHVD) in Garmian, Kurdistan region of Iraq: A clinical and molecular study.

INTRODUCTION: Koi herpesvirus disease (KHVD) is attributed to cyprinid herpesvirus-3 (CyHV-3) and predominantly affects common carp and ornamental koi carp (Cyprinus carpio). This viral infection leads to substantial morbidity and mortality among these fish species. This study aimed to confirm the presence of KHVD in the Kurdistan region of Iraq by employing clinical and optimized molecular assays on fish populations experiencing high mortality among common carp in carp farms. METHODOLOGY: The present research was conducted in the Kalar district, situated at the heart of Garmian province in Iraqi Kurdistan. four samples from common carp fish farms were received by our laboratory. These samples specifically displaying clinical signs associated with koi herpesvirus (KHV) infection, were subjected to clinical examinations, and PCR assay in addition to sequence analysis. RESULTS: The results of the current study revealed that the observed clinical signs, particularly gill necrosis, skin lesions, and sunken eyes, closely resembled the clinical signs of KHVD in common carp fish. In addition, PCR, nested PCR, and sequence analysis assay detected appropriate DNA fragments of the CyHV-3 major capsid protein gene confirming the first detection of KHVD in common carp fish in the Kurdistan region of Iraq. CONCLUSION: In this study, the results confirm the detection of KHVD in the Kurdistan region, Iraq, for the first time. This study revealed that CyHV-3 was responsible for KHVD-related signs and symptoms. Based on these results, it is strongly recommended that comprehensive studies be initiated to investigate the prevalence and distribution of CyHV-3.

Alzheimer's disease and herpes viruses: Current events and perspectives.

Alzheimer's disease (AD) is a real and current scientific and societal challenge. Alzheimer's disease is characterised by a neurodegenerative neuroinflammatory process, but the etiopathogenetic mechanisms are still unclear. The possible infectious aetiology and potential involvement of Herpes viruses as triggers for the formation of extracellular deposits of amyloid beta (Aß) peptide (amyloid plaques) and intraneuronal aggregates of hyperphosphorylated and misfold could be a possible explanation. In fact, the possible genetic interference of Herpes viruses with the genome of the host neuronal cell or the stimulation of the infection to a continuous immune response with a consequent chronic inflammation could constitute those mechanisms underlying the development of AD, with possible implications in the understanding and management of the disease. Herpes viruses could be significantly involved in the pathogenesis of AD and in particular, their ability to reactivate in particular conditions such as immunocompromise and immunosenescence, could explain the neurological damage characteristic of AD. Our review aims to evaluate the state of the art of knowledge and perspectives regarding the potential relationship between Herpes viruses and AD, in order to be able to identify the possible etiopathogenetic mechanisms and the possible therapeutic implications.

Development of multi epitope subunit vaccines against emerging carp viruses Cyprinid herpesvirus 1 and 3 using immunoinformatics approach.

Cyprinid herpesvirus is a causative agent of a destructive disease in common and koi carp (Cyprinus carpio), which leads to substantial global financial losses in aquaculture industries. Among the strains of C. herpesvirus, C. herpesvirus 1 (CyHV-1) and C. herpesvirus 3 (CyHV-3) are known as highly pathogenic to carp fishes in Europe, Asia, and Africa. To date, no effective vaccine has been developed to combat these viruses. This study aimed to develop unique multi-epitope subunit vaccines targeting the CyHV-1 and CyHV-3 using a reverse vaccinology approach. The study began with a comprehensive literature review to identify the most critical proteins, which were then subjected to in silico analyses to predict highly antigenic epitopes. These analyses involved assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. We constructed two multi-epitope-based vaccines incorporating a suitable adjuvant and appropriate linkers. It revealed that both the vaccines are non-toxic and immunogenic. The tertiary structures of the vaccine proteins were generated, refined, and validated to ensure their suitability. The binding affinity between the vaccine constructs and TLR3 and TLR5 receptors were assessed by molecular docking studies. Molecular dynamics simulations indicated that vaccine construct V1 exhibited greater stability with both TLR3 and TLR5 based on RMSD analysis. Hydrogen bond analysis revealed a stronger binding affinity between the vaccine constructs and TLR5 compared to TLR3. Furthermore, MM-PBSA analysis suggested that both vaccine constructs exhibited a better affinity for TLR5. Considering all aspects, the results suggest that in silico development of CyHV vaccines incorporating multiple epitopes holds promise for management of diseases caused by CyHV-1 and CyHV-3. However, further in vivo trials are highly recommended to validate the efficacies of these vaccines.

The Terminase Complex of Each Human Herpesvirus.

The human herpesviruses (HHVs) are classified into the following three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. These HHVs have distinct pathological features, while containing a highly conserved viral replication pathway. Among HHVs, the basic viral particle structure and the sequential processes of viral replication are nearly identical. In particular, the capsid formation mechanism has been proposed to be highly similar among herpesviruses, because the viral capsid-organizing proteins are highly conserved at the structural and functional levels. Herpesviruses form capsids containing the viral genome in the nucleus of infected cells during the lytic phase, and release infectious virus (i.e., virions) to the cell exterior. In the capsid formation process, a single-unit-length viral genome is encapsidated into a preformed capsid. The single-unit-length viral genome is produced by cleavage from a viral genome precursor in which multiple unit-length viral genomes are tandemly linked. This encapsidation and cleavage is carried out by the terminase complex, which is composed of viral proteins. Since the terminase complex-mediated encapsidation and cleavage is a virus-specific mechanism that does not exist in humans, it may be an excellent inhibitory target for anti-viral drugs with high virus specificity. This review provides an overview of the functions of the terminase complexes of HHVs.

Immunostimulation of Asian elephant (Elephas maximus) blood cells by parapoxvirus ovis and CpG motif-containing bacterial plasmid DNA upregulates innate immune gene expression.

The immune system of Asian elephants (Elephas maximus) is poorly studied, compared to that of livestock, rodents or humans. The innate immune response has become a focus of interest in relation to Elephant endotheliotropic herpesviruses (EEHVs). EEHVs cause a fatal hemorrhagic disease (EEHV-HD) and are a significant threat to captive Asian elephant populations worldwide. Similar to other herpesvirus infections, nearly all animals become infected, but only some develop disease. As progression to EEHV-HD is often acute, a robust innate immune response is crucial to control EEHV infections. This is invariably true of the host in the first instance, but it can also potentially be modulated by intervention strategies. Here, two immunostimulant veterinary medicinal products, authorized for use in domestic species, were tested for their ability to induce innate anti-viral immune responses in Asian elephant blood cells. Sequence data were obtained for a range of previously unidentified Asian elephant immune genes, including C-X-C motif chemokine ligand 10 (CXCL10), interferon stimulated gene 15 (ISG15) and myxovirus GTPase 1 (Mx1), and were employed in the design of species-specific qPCR assays. These assays were subsequently used in analyses to determine fold changes in gene expression over a period of 24 hours. This study demonstrates that both immunostimulant medications are capable of inducing significant innate anti-viral immune responses which suggests that both could be beneficial in controlling EEHV infections in Asian elephants.

Molecular characterization of equine herpes viruses type 1 and 4 among Arabian horse populations in Egypt during the period between 2021 and 2022.

Background: Equine herpesvirus type 1 (EHV-1) is a major cause of abortion and respiratory disease. Equine herpesvirus type 4 (EHV-4), on the other hand, is exclusively associated with respiratory disease in horse populations worldwide, particularly in Egypt and Arabian countries. Aim: This study aims to investigate the circulation of EHV-1 and EHV-4 in the Arabian horse population through molecular detection and genetic characterization of EHV-1 and/or EHV-4 that may threaten the stability of horse industry. Methods: A total of 80 samples including 50 nasal swabs, 10 vaginal swabs and 20 whole blood samples were collected from vaccinated and registered pure-bred Arabian adult horses from different studs in the governorates of northern Egypt (Cairo, Dakahlyia and Qalyubia) from 2021 to 2022. The collected samples were screened using consensus PCR for detection of EHVs using specific primers targeting DNA polymerase gene. The positive samples were subjected to conventional PCR for detection of EHV-1 and/or EHV-4using specific primers targeting glycoprotein (gB) gene. EHV-1 and EHV-4 amplicons were partially sequenced and phylogenetically analyzed using Sanger method. Results: Consensus PCR revealed that 48 out of 80 samples were positive for EHVs with percentage of 60%. Typing of the selected positive samples using conventional PCR showed that 29 out of 80 were positive for EHV-1 with percentage 36.25%, while 24 out of 80 samples were positive for EHV-4 with percentage 30%. Mixed infections with both viruses were detected in five samples. The amplified products were sequenced using Sanger method and submitted to GenBank under accession number OM362231MG-1 for EHV-1 strain and OM362232 MG-4 for EHV-4 strain. Sequence analysis and alignments of the amplified fragments of the EHV-1 and EHV-4 glycoprotein B (gB) gene to that of GenBank-derived reference strains revealed a high degree of similarity. According to the phylogenetic tree, the obtained sequences of EHV-1 and 4 in the current study showed homogeneity with local Egyptian and foreign EHV-1 and 4 strains and heterogeneity with EHV-2 and 5. Conclusion: The current investigation showed that molecular methods are appropriate assays for an efficient and accurate diagnosis of EHVs. Furthermore, it supports earlier research findings about the prevalence of EHV-1 and 4 in Arabian horse populations in Egypt.

Pseudorabies virus gM and its homologous proteins in herpesviruses induce mitochondria-related apoptosis involved in viral pathogenicity.

Apoptosis is a critical host antiviral defense mechanism. But many viruses have evolved multiple strategies to manipulate apoptosis and escape host antiviral immune responses. Herpesvirus infection regulated apoptosis; however, the underlying molecular mechanisms have not yet been fully elucidated. Hence, the present study aimed to study the relationship between herpesvirus infection and apoptosis in vitro and in vivo using the pseudorabies virus (PRV) as the model virus. We found that mitochondria-dependent apoptosis was induced by PRV gM, a late protein encoded by PRV UL10, a virulence-related gene involved in enhancing PRV pathogenicity. Mechanistically, gM competitively combines with BCL-XL to disrupt the BCL-XL-BAK complex, resulting in BCL-2-antagonistic killer (BAK) oligomerization and BCL-2-associated X (BAX) activation, which destroys the mitochondrial membrane potential and activates caspase-3/7 to trigger apoptosis. Interestingly, similar apoptotic mechanisms were observed in other herpesviruses (Herpes Simplex Virus-1 [HSV-1], human cytomegalovirus [HCMV], Equine herpesvirus-1 [EHV-1], and varicella-zoster virus [VZV]) driven by PRV gM homologs. Compared with their parental viruses, the pathogenicity of PRV-ΔUL10 or HSV-1-ΔUL10 in mice was reduced with lower apoptosis and viral replication, illustrating that UL10 is a key virulence-related gene in PRV and HSV-1. Consistently, caspase-3 deletion also diminished the replication and pathogenicity of PRV and HSV-1 in vitro and in mice, suggesting that caspase-3-mediated apoptosis is closely related to the replication and pathogenicity of PRV and HSV-1. Overall, our findings firstly reveal the mechanism by which PRV gM and its homologs in several herpesviruses regulate apoptosis to enhance the viral replication and pathogenicity, and the relationship between gM-mediated apoptosis and herpesvirus pathogenicity suggests a promising approach for developing attenuated live vaccines and therapy for herpesvirus-related diseases.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish.

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

Whole-genome assembly of a novel invertebrate herpesvirus from the gastropod Babylonia areolata.

Molluscan herpesviruses cause disease in species of major importance to aquaculture and are the only known herpesviruses to infect invertebrates, which lack an adaptive immune system. Understanding the evolution of malacoherpesviruses in relation to their hosts will likely require comparative genomic studies on multiple phylogenetic scales. Currently, only two malacoherpesvirus species have genomes that have been fully assembled, which limits the ability to perform comparative genomic studies on this family of viruses. In the present study, we fully assemble a herpesvirus from Illumina and Nanopore sequence data that were previously used to assemble the genome of the gastropod Babylonia areolata. We tentatively assign this novel herpesvirus to the genus Aurivirus within the family Malacoherpesviridae based on a phylogenetic analysis of DNA polymerase. While structurally similar to other malacoherpesvirus genomes, a synteny analysis of the novel herpesvirus with another Aurivirus species indicates that genomic rearrangements might be an important process in the evolution of this genus. We anticipate that future complete assemblies of malacoherpesviruses will be a valuable resource in comparative herpesvirus research.

Development and analytical validation of a novel quantitative PCR assay for the detection of Trachemys herpesvirus 1.

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 107 to 1.0 × 101 copies per reaction with an R2 of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 101 copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.

Herpesvirus Infection of Endothelial Cells as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Understanding the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that a herpesvirus infection of endothelial cells (ECs) may underlie ME/CFS symptomatology. We review evidence linking herpesviruses to persistent EC infection and the implications for endothelial dysfunction, encompassing blood flow regulation, coagulation, and cognitive impairment-symptoms consistent with ME/CFS and Long COVID. This paper provides a synthesis of current research on herpesvirus latency and reactivation, detailing the impact on ECs and subsequent systemic complications, including latent modulation and long-term maladaptation. We suggest that the chronicity of ME/CFS symptoms and the multisystemic nature of the disease may be partly attributable to herpesvirus-induced endothelial maladaptation. Our conclusions underscore the necessity for further investigation into the prevalence and load of herpesvirus infection within the ECs of ME/CFS patients. This review offers conceptual advances by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research toward potentially unexplored avenues in understanding and treating this complex syndrome.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay.

BACKGROUND: Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. RESULTS: The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10- 1 copies/µL for the FHV-1 TK gene and 5.5 copies/µL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. CONCLUSIONS: The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.

SELECTED INSTANCES OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS SHEDDING IN TRUNK SECRETIONS BY AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN COMPARISON TO SHEDDING BY ASIAN ELEPHANTS (ELEPHAS MAXIMUS).

This study examined the viral shedding kinetics of elephant endotheliotropic herpesvirus (EEHV) in African elephants (Loxodonta africana) compared to viral shedding behavior in Asian elephants (Elephas maximus). Little is known about the transmission dynamics and epidemiology of this disease in African elephants. In light of recent clinical cases and mortalities, this paper aims to identify trends in viral biology. Trunk wash samples were collected from 22 African elephants from four North American zoological institutions that had recently experienced herd viremias or translocations. Processing of these samples included DNA extraction followed by qPCR to quantitate viral DNA load. The results were then compared with available literature that chronicled similar cases in Asian and African elephants. Minimal EEHV shedding was detected in response to varied herd translocations. Increased shedding was recorded in herds in which an elephant experienced an EEHV viremia when compared to baseline shedding. These index infections were followed by subsequent viremias in other elephants, although it is not known if these were recrudescence, transient controlled viremias, and/or primary infections via transmission to other elephants. When compared to historically published data, it was observed that EEHV3 cases in African elephants and EEHV1A cases in Asian elephants had consistently higher levels of viral DNA in the blood than were shed in trunk secretions, a fact that is seemingly inconsistent with such severe cases of disease and the high mortality rates associated with those respective types. The findings produced in this study highlight the need for more routine monitoring of viral shedding in African elephant herds to elucidate possible EEHV transmission and recrudescence factors for ex situ population management.

INVESTIGATION OF A POINT-OF-CARE VISCOELASTIC COAGULATION MONITOR AND ITS COMPARISON TO THROMBOELASTOGRAPHY IN CLINICALLY HEALTHY AFRICAN ELEPHANTS (LOXODONTA AFRICANA).

Elephant endotheliotropic herpesvirus (EEHV) can induce fatal hemorrhagic disease (HD) in African elephants (Loxodonta africana). Once clinical signs develop, progression is rapid, even with aggressive treatment. There is a critical need to develop point-of-care diagnostic tests to aid in identification of EEHV-HD prior to the onset of overt clinical signs. Study objectives were to investigate a novel, point-of-care viscoelastic coagulation monitor (VCM Vet), compare the results to thromboelastography (TEG), and report traditional hemostatic analytes in adult African elephants. Whole blood was collected from seven clinically healthy elephants (four females and three males, 18-47 yr) and analyzed in duplicate via VCM Vet and kaolin-activated TEG 1-3 and 30 min following collection, respectively. Separated plasma was frozen for ancillary coagulation testing. Both analyses generated quantifiable clotting reactions with variables (median [range]) describing clot formation rate (VCM Vet, clot time = 682 s [530-987 s], clot formation time = 244 s [186-744 s], Alpha = 40° [14-47°]; TEG, reaction time = 6.2 min [3.7-11.8 min], kinetic time = 1.3 min [0.9-2.6 min], Alpha = 70° [57-77°]), clot strength (VCM Vet, maximum clot formation = 34 units [20-45 units]; TEG, maximum amplitude = 75 mm [69-80 mm], shear elastic modulus strength = 14.7 Kdynes/s [11.3-19.5 Kdynes/s]), and clot lysis (VCM Vet, lysis index at 30 min = 100% [100-99%], lysis index at 45 min = 98% [95-100%]; TEG, lysis index at 30 min = 0% [0-0.4%], lysis index at 60 min = 1.4% [0-2.6%]) recorded. Additional testing (median [range]) included D-dimer concentration (33 ng/ml [28-94 ng/ml]), prothrombin time (12.4 s [12.2-13.2 s]), activated partial thromboplastin time (17.2 s [14.2-18.8 s]), and fibrinogen concentration (297 [282-383] mg/dL). Tracings generated by VCM Vet and TEG were clinically similar, and there was visual agreement and minimal difference between quantitative variables for duplicate tests. VCM Vet is a promising, user-friendly tool for use in identification and management of coagulopathies in African elephants.

FIRST DETECTION OF CLINICAL DISEASE DUE TO ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 7A IN TWO AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN HUMAN CARE.

Multiple species of elephant endotheliotropic herpesvirus (EEHV) have caused fatal hemorrhagic disease in African (Loxodonta africana) and Asian (Elephas maximus) elephants. To date, EEHV7 has been detected only in benign pulmonary and skin nodules and in saliva of African elephants and has not been associated with clinical illness. Low-level viremia due to EEHV7A was detected via qPCR in two subadult African elephants during routine surveillance. Hematologic changes were noted in both elephants, including leukopenia, lymphopenia, monocytopenia, and band heterophilia. Treatment was initiated with famciclovir, antimicrobials, and rectal fluids, and one elephant received plasma transfusions due to a progressive decrease in platelet count. Both elephants remained asymptomatic throughout the viremias, with rapid resolution of hematologic abnormalities. These cases add to the current understanding of the epidemiology of EEHV in African elephants; to the authors' knowledge, they represent the first documentation of clinical disease due to EEHV7 infection in any elephant.

Otariid gammaherpesvirus 1 in South American fur seals (Arctocephalus australis) and a novel related herpesvirus in free-ranging South American sea lions (Otaria byronia): Prevalence and effects of age, sex, and sample type.

Otariid gammaherpesvirus 1 (OtGHV1) is associated with high rates of urogenital carcinoma in free-ranging California sea lions (Zalophus californianus; CSL), and until recently was reported only in the Northern Hemisphere. The objective of this study was to survey free-ranging South American sea lions (Otaria byronia; SASL) and South American fur seals (Arctocephalus australis: SAFS) in Punta San Juan, Peru for OtGHV1 and to determine prevalence characteristics. Twenty-one percent (14/67) of urogenital swabs collected over three years (2011, 2014, 2015) from live pinnipeds of both species tested positive with a pan-herpesvirus conventional PCR. Sequencing of SAFS amplicons revealed 100% homology to OtGHV1 at the DNA polymerase, glycoprotein B, and viral bcl2-like genes. Sequencing of SASL amplicons revealed a novel related virus, herein called Otariid gammaherpesvirus 8 (OtGHV8). For comparison of sample sites, urogenital, conjunctival, and oropharyngeal swabs collected from 136 live pinnipeds of both species at Punta San Juan between 2011-2018 were then assayed using quantitative PCR for a segment of the OtGHV1/8 DNA polymerase gene using a qPCR assay now determined to cross-react between the two viruses. In total, across both species, 38.6% (51/132) of urogenital swabs, 5.6% (4/71) of conjunctival swabs, and 1.1% (1/90) of oropharyngeal swabs were positive for OtGHV1/8, with SASL only positive on urogenital swabs. Results from SASL were complicated by the finding of OtGHV8, necessitating further study to determine prevalence of OtGHV1 versus OtGHV8 using an alternate assay. Results from SAFS suggest a potential relationship between OtGHV1 in SAFS and CSL. Though necropsy surveillance in SAFS is very limited, geographic patterns of OtGHV1-associated urogenital carcinoma in CSL and the tendency of herpesviruses to cause more detrimental disease in aberrant hosts suggests that it is possible that SAFS may be the definitive host of OtGHV1, which gives further insight into the diversity and phyogeography of this clade of related gammaherpesviruses.