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.

First molecular confirmation of cyprinid herpesvirus 1 (CyHV1) in diseased carp in Serbia.

Mass mortality of farmed 1 yr old common carp Cyprinus carpio occurred at a carp farm in April 2022. In addition to high mortality, diseased fish exhibited papillomatous growths on the skin and fins, characteristic of carp pox. To investigate a possible viral cause, tissue samples were collected and nucleic acid was extracted using standard procedures. In a pooled sample from the gills and kidneys, carp edema virus (CEV) was detected by real-time PCR. In a skin tissue sample with papillomatous growths, cyprinid herpesvirus 1 (CyHV1) was detected by a conventional PCR targeting a conserved region of the DNA polymerase of cyprinid herpesviruses. PCR products were visualized through agarose gel electrophoresis, and the presence of CyHV1 DNA was confirmed by Sanger sequencing. This represents the first molecular confirmation of CyHV1 in common carp in Serbia.

Susceptibility of shellfish aquaculture species in the Chesapeake Bay and Maryland coastal bays to ostreid herpesvirus 1 microvariants.

Ostreid herpesvirus 1 (OsHV-1) and its microvariants (µVars) cause economically devastating mass mortalities of oysters and pose a threat to the shellfish aquaculture industry globally. OsHV-1 outbreaks can cause up to 100% mortality in the Pacific oyster Crassostrea gigas. However, OsHV-1 and its variants have a broad host range and can infect at least 7 bivalve species, including bay scallops Argopecten irradians and eastern oysters C. virginica. Determining the susceptibility of economically and ecologically important bivalve species to OsHV-1 is critical for improving biosecurity and disease management to protect the aquaculture industry. Surveys of eastern oysters were conducted in June to August 2021 in the Maryland portion of the Chesapeake Bay to determine the prevalence and viral load of OsHV-1 at 5 aquaculture farms. Using quantitative PCR, OsHV-1 was not detected at any sites. Experiments examined the susceptibility of single stocks of eastern oysters and hard clams Mercenaria mercenaria to the virus and their ability to horizontally transmit it using OsHV-1 µVar SD (San Diego, California) and OsHV-1 µVar FRA (Marennes-Olreon, France). Results showed that OsHV-1 µVars did not cause mortality or symptomatic infection in the single stocks of eastern oysters and hard clams used in these experiments using natural infection pathways. However, the eastern oyster stock, when injected with OsHV-1, did transmit the virus to naïve Pacific oysters. Further experimentation using additional stocks and lines and establishment of surveillance programs along the east and Gulf coasts of the USA are necessary to prepare for the potential spread and impact of OsHV-1 related disease.

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.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

BIOLOGICAL VARIATION OF HEMATOLOGY PARAMETERS AND CLINICAL APPLICATION IN AFRICAN ELEPHANTS (LOXODONTA AFRICANA).

Detailed knowledge of biological variation can facilitate accurate interpretation of clinical pathology parameters. A recent biological variation study in Asian elephants (Elephas maximus) found that hematology parameters had high individuality, which suggests that population-derived reference intervals may be an insensitive diagnostic tool. In elephant medicine, sensitive hematology-related diagnostics are crucial for clinical decision-making, particularly in elephants at risk for elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD). The objective of this study was to assess biological variation of hematology parameters in African elephants to determine whether population-derived reference intervals are a sensitive diagnostic tool for interpreting results and to provide a useful alternative. Eight healthy African elephants had blood collected under behavioral training every other week for 8 wk. Complete blood cell count (CBC) analysis was performed in duplicate to assess analytical variation. Previous methods were used to determine between-individual variation, within-individual variation, index of individuality, and reference change values (RCV). This study found that most hematology parameters displayed intermediate-to-high individuality, which suggests that alternatives to population-derived reference intervals are necessary to detect pathologic changes. To test the results of our biological variation data, a case of EEHV-HD was retrospectively evaluated. Individual normal values and calculated RCV detected a clinically significant monocytopenia, leukopenia, and thrombocytopenia associated with EEHV2 viremia. However, none of these parameters fell outside a population-derived reference interval. This study highlights the utility of biological variation in clinical decision-making and demonstrates that individual normal values and RCV may be important diagnostic tools for CBC interpretation in African elephants.

'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.

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.

Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication.

Pseudorabies is an acute and febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3-3937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.

Transcriptomic Responses to Koi Herpesvirus in Isolated Blood Leukocytes from Infected Common Carp.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.

Acipenserid herpesvirus 2 genome and partial validation of a qPCR for its detection in white sturgeon Acipenser transmontanus.

White sturgeon Acipenser transmontanus is the primary species used for caviar and sturgeon meat production in the USA. An important pathogen of white sturgeon is acipenserid herpesvirus 2 (AciHV-2). In this study, 4 archived isolates from temporally discrete natural outbreaks spanning the past 30 yr were sequenced via Illumina and Oxford Nanopore Technologies platforms. Assemblies of approximately 134 kb were obtained for each isolate, and the putative ATPase subunit of the terminase gene was selected as a potential quantitative PCR (qPCR) target based on sequence conservation among AciHV-2 isolates and low sequence homology with other important viral pathogens. The qPCR was repeatable and reproducible, with a linear dynamic range covering 5 orders of magnitude, an efficiency of approximately 96%, an R2 of 0.9872, and an analytical sensitivity of 103 copies per reaction after 35 cycles. There was no cross-reaction with other known viruses or closely related sturgeon species, and no inhibition by sturgeon DNA. Clinical accuracy was assessed from white sturgeon juveniles exposed to AciHV-2 by immersion. Viral culture (gold standard) and qPCR were in complete agreement for both cell culture negative and cell culture positive samples, indicating that this assay has 100% relative accuracy compared to cell culture during an active outbreak. The availability of a whole-genome sequence for AciHV-2 and a highly specific and sensitive qPCR assay for detection of AciHV-2 in white sturgeon lays a foundation for further studies on host-pathogen interactions while providing a specific and rapid test for AciHV-2 in captive and wild populations.

Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation.

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Survey of selected viral agents (herpesvirus, adenovirus and hepatitis E virus) in liver and lung samples of cetaceans, Brazil.

Hepatic and pulmonary lesions are common in cetaceans, despite their poorly understood viral etiology. Herpesviruses (HV), adenoviruses (AdV) and hepatitis E virus (HEV) are emerging agents in cetaceans, associated with liver and/or pulmonary damage in mammals. We isolated and molecularly tested DNA for HV and AdV (n = 218 individuals; 187 liver and 108 lung samples) and RNA for HEV (n = 147 animals; 147 liver samples) from six cetacean families. All animals stranded or were bycaught in Brazil between 2001 and 2021. Positive-animals were analyzed by histopathology. Statistical analyses assessed if the prevalence of viral infection could be associated with the variables: species, family, habitat, region, sex, and age group. All samples were negative for AdV and HEV. Overall, 8.7% (19/218) of the cetaceans were HV-positive (4.8% [9/187] liver and 11.1% [12/108] lung), without HV-associated lesions. HV-prevalence was statistically significant higher in Pontoporiidae (19.2%, 10/52) when compared to Delphinidae (4.1%, 5/121), and in southeastern (17.1%, 13/76)-the most industrialized Brazilian region-when compared to the northeastern region (2.4%, 3/126). This study broadens the herpesvirus host range in cetaceans, including its description in pygmy sperm whales (Kogia breviceps) and humpback whales (Megaptera novaeangliae). Further studies must elucidate herpesvirus drivers in cetaceans.

Diagnostic performance of cross-priming amplification-based lateral flow assay (CPA-LFA) and real-time PCR for koi herpesvirus (KHV) detection.

Epizootics of Koi herpesvirus (KHV) cause mass mortality in koi carp (Cyprinus rubrofuscus) and common carp (Cyprinus carpio) worldwide. Rapid and accurate virus detection technology is crucial for preventing pathogen spread and minimizing damage. Although several diagnostic assays have been developed for KHV, the analytical and diagnostic performance of the detection methods has not been evaluated. In this study, we developed and validated the diagnostic performance of two molecular diagnostic assays, cross-priming amplification-based lateral flow assay (CPA-LFA) and TaqMan probe-based real-time polymerase chain reaction (PCR). To detect KHV, primers and probe were designed based on the thymidine kinase (TK) genes. The detection limits of developed CPA-LFA and real-time PCR assays were determined to be 675.69 copies/µL and 8.384 copies/µL, respectively. The diagnostic sensitivity and specificity of the developed assay were determined using fish samples (n = 179). CPA-LFA was found to be 93.67% and 100%, respectively, and real-time PCR was found to be 100% and 100%, respectively. Therefore, the newly developed CPA-LFA and real-time PCR assays accurately and rapidly detect KHV. CPA-LFA is particularly suitable for point-of-care diagnosis because of its simple diagnostic process, and real-time PCR analysis is most suitable for precise diagnosis because it can detect low viral loads.

Low gH/gL (Sub)Species-Specific Antibody Levels Indicate Elephants at Risk of Fatal Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease.

Elephant endotheliotropic herpesviruses (EEHVs), of which eleven (sub)species are currently distinguished, infect either Asian (Elephas maximus) or African elephants (Loxodonta species). While all adult elephants are latently infected with at least one EEHV (sub)species, young elephants, specifically those with low to non-detectable EEHV-specific antibody levels, may develop fatal hemorrhagic disease (EEHV-HD) upon infection. However, animals with high antibody levels against EEHV(1A) gB, an immunodominant antigen recognized by antibodies elicited against multiple (sub)species, may also occasionally succumb to EEHV-HD. To better define which animals are at risk of EEHV-HD, gB and gH/gL ELISAs were developed for each of the Asian elephant EEHV subspecies and assessed using 396 sera from 164 Asian elephants from European zoos. Antibody levels measured against gB of different (sub)species correlated strongly with one another, suggesting high cross-reactivity. Antibody levels against gH/gL of different subspecies were far less correlated and allowed differentiation between these (sub)species. Importantly, while high gB-specific antibody levels were detected in the sera of several EEHV-HD fatalities, all fatalities (n = 23) had low antibody levels against gH/gL of the subspecies causing disease. Overall, our data indicate that (sub)species-specific gH/gL ELISAs can be used to identify animals at risk of EEHV-HD when infected with a particular EEHV (sub)species.