Intragenic MFSD8 duplication and histopathological findings in a rabbit with neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCL) are among the most prevalent neurodegenerative disorders of early life in humans. Disease-causing variants have been described for 13 different NCL genes. In this study, a refined pathological characterization of a female rabbit with progressive neurological signs reminiscent of NCL was performed. Cytoplasmic pigment present in neurons was weakly positive with Sudan black B and autofluorescent. Immunohistology revealed astrogliosis, microgliosis and axonal degeneration. During the subsequent genetic investigation, the genome of the affected rabbit was sequenced and examined for private variants in NCL candidate genes. The analysis revealed a homozygous ~10.7 kb genomic duplication on chromosome 15 comprising parts of the MFSD8 gene, NC_013683.1:g.103,727,963_103,738,667dup. The duplication harbors two internal protein coding exons and is predicted to introduce a premature stop codon into the transcript, truncating ~50% of the wild-type MFSD8 open reading frame encoding the major facilitator superfamily domain containing protein 8, XP_002717309.2:p.(Glu235Leufs*23). Biallelic loss-of-function variants in MFSD8 have been described to cause NCL7 in human patients, dogs and a single cat. The available clinical and pathological data, together with current knowledge about MFSD8 variants and their functional impact in other species, point to the MFSD8 duplication as a likely causative defect for the observed phenotype in the affected rabbit.

Pathological findings in South American camelids presented at a farm animal clinic in Northern Germany (2005-2021).

To expand the knowledge about common diseases in llamas and alpacas in Germany, a screening of the cases of South American camelids presented at the Clinic for Swine and Small Ruminants of the University of Veterinary Medicine Hannover, Germany from 2005 to the end of November 2021 was performed. A retrospective evaluation of necropsy reports from this period was conducted. Overall, necropsy reports were evaluated from 187 alpacas, 35 llamas and one vicuña (n = 223). A total of 50.2% of the dissected animals were thin or cachectic. Pathological alterations of the gastrointestinal tract were the most common findings (44.8%). In addition, liver changes were recorded, most frequently in adult animals. In contrast, diseases of the respiratory tract and the nervous system were found more frequently in juvenile animals. This study provides an overview of common pathologies in South American camelids in Germany and thus may help to recognise different disease symptoms at an early stage.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

RALGAPA1 Deletion in Belgian Shepherd Dogs with Cerebellar Ataxia.

Several genetically distinct forms of cerebellar ataxia exist in Belgian shepherd dogs. We investigated a litter in which two puppies developed cerebellar ataxia. The clinical signs stabilized at around six weeks of age, but remained visible into adulthood. Combined linkage and homozygosity mapping delineated a 5.5 Mb critical interval. The comparison of whole-genome sequence data of one affected dog to 929 control genomes revealed a private homozygous ~4.8 kb deletion in the critical interval, Chr8:14,468,376_14,473,136del4761. The deletion comprises exon 35 of the RALGAPA1 gene, XM_038544497.1:c.6080-2893_6944+1003del. It is predicted to introduce a premature stop codon into the transcript, truncating ~23% of the wild-type open reading frame of the encoded Ral GTPase-activating protein catalytic subunit α 1, XP_038400425.1:(p.Val2027Glnfs*7). Genotypes at the deletion showed the expected co-segregation with the phenotype in the family. Genotyping additional ataxic Belgian shepherd dogs revealed three additional homozygous mutant dogs from a single litter, which had been euthanized at five weeks of age due to their severe clinical phenotype. Histopathology revealed cytoplasmic accumulation of granular material within cerebellar Purkinje cells. Genotyping a cohort of almost 900 Belgian shepherd dogs showed the expected genotype-phenotype association and a carrier frequency of 5% in the population. Human patients with loss-of-function variants in RALGAPA1 develop psychomotor disability and early-onset epilepsy. The available clinical and histopathological data, together with current knowledge about RALGAPA1 variants and their functional impact in other species, suggest the RALGAPA1 deletion is the likely causative defect for the observed phenotype in the affected dogs.

Case report: Canine distemper virus infection as a cause of central nervous system disease in a Eurasian lynx (Lynx lynx).

The Eurasian lynx (Lynx lynx) represents an endangered species with only small populations remaining in Central Europe. Knowledge about the threat posed by potential infectious agents to these animals is crucial for informing ongoing protection measures. Canine distemper virus (CDV) is known to have a wide host range with infection reported in many mammalian species including several lynx species (Lynx pardinus, Lynx canadensis, Lynx rufus), but is an extremely rare finding in the Eurasian lynx. The present report describes a case of a Eurasian lynx showing central nervous signs, including apathy and ataxia. A CT scan revealed multiple hypodense areas in different localizations within the brain as well as enlarged liquid filled areas, leading to the suspicion of a degenerative process. Due to clinical deterioration, the animal was euthanized and submitted for macroscopical and histological investigations. Histological investigations revealed multifocal demyelinations in the cerebellum, brain stem and cervical spinal cord as well as a multifocal, perivascular, lymphohistiocytic meningoencephalitis. A CDV infection was confirmed by immunohistochemistry and RT-PCR analyses. This CDV infection of a Eurasian lynx resembles a classical chronic manifestation of distemper in dogs and highlights the threat posed by canine distemper to this species.

Functional Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Delivered by Canine Histiocytic Sarcoma Cells Persistently Infected with Engineered Attenuated Canine Distemper Virus.

The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte-macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.

Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice.

Introduction: Tick-borne encephalitis virus (TBEV) is one of the most relevant tick-transmitted neurotropic arboviruses in Europe and Asia and the causative agent of tick-borne encephalitis (TBE). Annually more than 10,000 TBE cases are reported despite having vaccines available. In Europe, the vaccines FSME-IMMUN® and Encepur® based on formaldehyde-inactivated whole viruses are licensed. However, demanding vaccination schedules contribute to sub-optimal vaccination uptake and breakthrough infections have been reported repeatedly. Due to its immunogenic properties as well as its role in viral replication and disease pathogenesis, the non-structural protein 1 (NS1) of flaviviruses has become of interest for non-virion based flavivirus vaccine candidates in recent years. Methods: Therefore, immunogenicity and protective efficacy of TBEV NS1 expressed by neuraminidase (NA)-deficient Influenza A virus (IAV) or Modified Vaccinia virus Ankara (MVA) vectors were investigated in this study. Results: With these recombinant viral vectors TBEV NS1-specific antibody and T cell responses were induced. Upon heterologous prime/boost regimens partial protection against lethal TBEV challenge infection was afforded in mice. Discussion: This supports the inclusion of NS1 as a vaccine component in next generation TBEV vaccines.

A recombinant Modified Vaccinia virus Ankara expressing prME of tick-borne encephalitis virus affords mice full protection against TBEV infection.

Introduction: Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause a serious disease involving the central nervous system (tick-borne encephalitis, TBE). Although approved inactivated vaccines are available, the number of TBE cases is rising, and breakthrough infections in fully vaccinated subjects have been reported in recent years. Methods: In the present study, we generated and characterized a recombinant Modified Vaccinia virus Ankara (MVA) for the delivery of the pre-membrane (prM) and envelope (E) proteins of TBEV (MVA-prME). Results: MVA-prME was tested in mice in comparison with a licensed vaccine FSME-IMMUN® and proved to be highly immunogenic and afforded full protection against challenge infection with TBEV. Discussion: Our data indicate that MVA-prME holds promise as an improved next-generation vaccine for the prevention of TBE.

Hypertensive nonobstructive hydrocephalus as main magnetic resonance imaging feature in a dog with disseminated choroid plexus carcinomatosis.

Obstructive or nonobstructive hypertensive hydrocephalus is reported in choroid plexus tumors. Choroid plexus tumors typically present as T2-weighted hyperintense intraventricular masses with occasional cerebrospinal fluid-drop metastasis. Acquired neoplastic nonobstructive hydrocephalus without visible mass lesion in magnetic resonance imaging is not reported in dogs. A 4.5-year-old Rhodesian Ridgeback presented with reduced mental status, unilaterally absent pupillary light reflex, and neck pain. Magnetic resonance imaging revealed a nonobstructive hydrocephalus and widened lumbar subarachnoid space with no evidence of a primary mass lesion. Postmortem examination confirmed a disseminated choroid plexus tumor affecting the ependyma and choroid plexi of all ventricles and the cerebral and lumbar subarachnoid space. Disseminated choroid plexus carcinomatosis should be considered as a possible cause of hypertensive hydrocephalus even in absence of a primary mass.

Rustrela Virus as Putative Cause of Nonsuppurative Meningoencephalitis in Lions.

Retrospective investigation of archived tissue samples from 3 lions displaying nonsuppurative meningoencephalitis and vasculitis led to the detection of rustrela virus (RusV). We confirmed RusV antigen and RNA in cortical neurons, axons, astrocytes and Purkinje cells by reverse transcription quantitative PCR, immunohistochemistry, and in situ hybridization.

Cross-reactive antibodies against Langat virus protect mice from lethal tick-borne encephalitis virus infection.

Introduction: Naturally attenuated Langat virus (LGTV) and highly pathogenic tick-borne encephalitis virus (TBEV) share antigenically similar viral proteins and are grouped together in the same flavivirus serocomplex. In the early 1970s, this has encouraged the usage of LGTV as a potential live attenuated vaccine against tick-borne encephalitis (TBE) until cases of encephalitis were reported among vaccinees. Previously, we have shown in a mouse model that immunity induced against LGTV protects mice against lethal TBEV challenge infection. However, the immune correlates of this protection have not been studied. Methods: We used the strategy of adoptive transfer of either serum or T cells from LGTV infected mice into naïve recipient mice and challenged them with lethal dose of TBEV. Results: We show that mouse infection with LGTV induced both cross-reactive antibodies and T cells against TBEV. To identify correlates of protection, Monitoring the disease progression in these mice for 16 days post infection, showed that serum from LGTV infected mice efficiently protected from developing severe disease. On the other hand, adoptive transfer of T cells from LGTV infected mice failed to provide protection. Histopathological investigation of infected brains suggested a possible role of microglia and T cells in inflammatory processes within the brain. Discussion: Our data provide key information regarding the immune correlates of protection induced by LGTV infection of mice which may help design better vaccines against TBEV.

Evidence for a Novel Gammaherpesvirus as the Putative Agent of Malignant Catarrhal Fever Disease in Roan Antelopes (Hippotragus equinus).

Upon the sudden death of two captive roan antelopes (Hippotragus equinus) that had suffered from clinical signs reminiscent of malignant catarrhal fever (MCF) in a German zoo, next generation sequencing of organ samples provided evidence of the presence of a novel gammaherpesvirus species. It shares 82.40% nucleotide identity with its so far closest relative Alcelaphine herpesvirus 1 (AlHV-1) at the polymerase gene level. The main histopathological finding consisted of lympho-histiocytic vasculitis of the pituitary rete mirabile. The MCF-like clinical presentation and pathology, combined with the detection of a nucleotide sequence related to that of AlHV-1, indicates a spillover event of a novel member of the genus Macavirus of the Gammaherpesvirinae, probably from a contact species within the zoo. We propose the name Alcelaphine herpesvirus 3 (AlHV-3) for this newly identified virus.

The modulating effect of food composition on the immune system in growing ring-necked pheasants (Phasianus colchicus).

The decline in the population of ring-necked pheasants (Phasianus colchicus) in northwestern Germany since 2007 raises questions about the underlying causes. We therefore studied the growth and immune status of ring-necked pheasant chicks dependent on different feed composition. Here, 490 ring-necked pheasant chicks were raised in five groups up to nine weeks. While control groups C1 and C2 received sufficient crude protein (28%) and energy (12.5 MJ/Kg feed) according to current standards, group C2 was treated with cyclosporine eight hours prior to phythemagglutination (PHA) testing, serving as a positive immune suppressed control. Group V1 was fed with reduced protein (20%) but optimal energy content (12.5 MJ/Kg feed), group V2 was fed with sufficient protein (28%) and reduced energy content (10 MJ/kg feed) whereas group V3 was fed reduced crude protein (20%) and reduced energy content (10MJ/kg feed). On all chicks, health status was checked each week, and 20 birds of each group were weighed randomly per week. PHA-testing was performed on 12 birds of each group to study the in vivo non-specific activation of lymphocytes at week 2, 4, 6, 7, 8 and 9. In addition, hemolysis-hemagglutination-assay (HHA) was performed on each of the PHA-tested chicks, which were subsequently euthanized and dissected. Histopathologic examinations of 5 birds that were randomly chosen were performed. The PHA-test results demonstrate significant differences between control (C1, C2) and experimental groups (V1-V3) in several developmental stages. According to the HHA results, weekly testing detected a significant increase of titres per week in all groups without significant differences. Here, only hemagglutination and no lysis of samples was observed. It seems appropriate to conclude that during their first weeks of life, protein content is of higher importance in ring-necked pheasant chicks than energy intake. In particular T-cell response is significantly reduced, which indicate a weaker immune system resulting in a higher risk for clinical diseases. Therefore, we assume that protein i.e. insect availability is a highly important co-factor in the free-ranging population dynamics, and is linked to declines of the northwestern German population.

Dose-Dependent Hepacivirus Infection Reveals Linkage between Infectious Dose and Immune Response.

More than 70 million people worldwide are still infected with the hepatitis C virus 30 years after its discovery, underscoring the need for a vaccine. To develop an effective prophylactic vaccine, detailed knowledge of the correlates of protection and an immunocompetent surrogate model are needed. In this study, we describe the minimum dose required for robust equine hepacivirus (EqHV) infection in equids and examined how this relates to duration of infection, seroconversion, and transcriptomic responses. To investigate mechanisms of hepaciviral persistence, immune response, and immune-mediated pathology, we inoculated eight EqHV naive horses with doses ranging from 1-2 copies to 1.3 × 106 RNA copies per inoculation. We characterized infection kinetics, pathology, and transcriptomic responses via next generation sequencing. The minimal infectious dose of EqHV in horses was estimated at 13 RNA copies, whereas 6 to 7 copies were insufficient to cause infection. Peak viremia did not correlate with infectious dose, while seroconversion and duration of infection appeared to be affected. Notably, seroconversion was undetectable in the low-dose infections within the surveillance period (40 to 50 days). In addition, transcriptomic analysis revealed a nearly dose-dependent effect, with greater immune activation and inflammatory response observed in high-dose infections than in low-dose infections. Interestingly, inoculation with 6-7 copies of RNA that did not result in productive infection, but was associated with a strong immune response, similar to that observed in the high-dose infections. IMPORTANCE We demonstrate that the EqHV dose of infection plays an important role for inducing immune responses, possibly linked to early clearance in high-dose and prolonged viremia in low-dose infections. In particular, pathways associated with innate and adaptive immune responses, as well as inflammatory responses, were more strongly upregulated in high-dose infections than in lower doses. Hence, inoculation with low doses may enable EqHV to evade strong immune responses in the early phase and therefore promote robust, long-lasting infection.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Gastric Ulcers in Alpacas- Clinical, Laboratory, and Pathological Findings.

Gastric ulcers are a common finding in post-mortem examinations of South American camelids (SAC), but diagnosis in living animals is often difficult. The aim of this study was to provide an overview of the incidence of gastric ulcers in alpacas, common concomitant diseases, and clinical as well as laboratory findings to facilitate diagnosis for veterinarians. For this purpose, a total of 187 necropsy reports of alpacas were evaluated, including clinical and laboratory findings on the living animal. A total of 23.5% of the animals (n = 44) were found to have gastric ulcers, nine were perforated. Compartment 3 was most frequently affected by gastric ulcers. No sex predilection could be detected, but animals 1 year of age and older were more frequently affected by gastric ulcers than animals under 1 year of age. Alpacas with gastric ulcers were presented to the clinic due to different non-specific symptoms. In alpacas with gastric ulcers, significantly more organs or organ systems besides the stomach revealed clinical findings than in animals without gastric ulcers. Of the 44 animals with gastric ulcers, a total of 21 alpacas (47.7%) had a poor nutritional status, but cachexia was not significantly more frequent in animals with gastric ulcers than in other dissected animals without ulcers. Hematologic investigations revealed a significantly lower white blood count and significantly lower segmented neutrophils than in deceased animals without ulcers. Compared to animals discharged after treatment, alpacas that died with gastric ulcers had significantly higher levels of band neutrophils and fewer eosinophils and basophils. Occult blood in feces was found in three of 12 animals with gastric ulcers examined for occult blood. In summary, gastric ulcers are a common problem in SAC, which is difficult to diagnose clinically or by laboratory investigations. As these are often chronic processes involving other organ systems, regular monitoring of the animals' nutritional status and early detection of disease symptoms may help to prevent gastric ulcers.

Persistent Infection of a Canine Histiocytic Sarcoma Cell Line with Attenuated Canine Distemper Virus Expressing Vasostatin or Granulocyte-Macrophage Colony-Stimulating Factor.

Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an in vitro characterization of two genetically engineered viruses based on the CDV strain Onderstepoort (CDV-Ond), CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF was performed. Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies.

[Eccrine adenoma in a cat]. / Adenom der ekkrinen Drüsen bei einer Katze.

Tumors originating from eccrine glands are rare findings in dogs and cats. In most cases, the tumors are malignant, while adenomas are only reported anecdotally. In the present case, a one-year-old female, spayed cat was presented with a swelling of the footpad of the right forelimb. Initially, the mass possessed a diameter of 2 cm which progressed to 4 cm within the following two months. At the latter time point the tumor was ulcerated. After surgical removal, histological and immunohistochemical analyses were performed. Histologically, a well demarcated, nodular, multilobular mass was present. The cuboidal to columnar neoplastic cells were arranged in tubular and acinar structures. Tumor cells possessed large, round to oval nuclei with moderately distinct nucleoli. Mitotic figures averaged 0-1 per high power field. Additionally, large areas of chondroid metaplasia were evident. Immunohistochemically, neoplastic cells were positive for pan-cytokeratin AE1/AE3 whereas thyroid transcription factor 1 (TTF1) was not expressed. Based on the histological and immunohistochemical findings an adenoma of the eccrine glands was diagnosed.

Experimental cross-species infection of donkeys with equine hepacivirus and analysis of host immune signatures.

BACKGROUND: The Equine Hepacivirus (EqHV) is an equine-specific and liver-tropic virus belonging to the diverse genus of Hepaciviruses. It was recently found in a large donkey (Equus asinus) cohort with a similar seroprevalence (30%), but lower rate of RNA-positive animals (0.3%) compared to horses. These rare infection events indicate either a lack of adaptation to the new host or a predominantly acute course of infection. METHODS: In order to analyze the susceptibility and the course of EqHV infection in donkeys, we inoculated two adult female donkeys and one control horse intravenously with purified EqHV from a naturally infected horse. Liver biopsies were taken before and after inoculation to study changes in the transcriptome. RESULTS: Infection kinetics were similar between the equids. All animals were EqHV PCR-positive from day three. EqHV RNA-levels declined when the animals seroconverted and both donkeys cleared the virus from the blood by week 12. Infection did not have an impact on the clinical findings and no significant histopathological differences were seen. Blood biochemistry revealed a mild increase in GLDH at the time of seroconversion in horses, which was less pronounced in donkeys. Transcriptomic analysis revealed a distinct set of differentially expressed genes, including viral host factors and immune genes. CONCLUSION: To summarize, our findings indicate that donkeys are a natural host of EqHV, due to the almost identical infection kinetics. The different immune responses do however suggest different mechanisms in reacting to hepaciviral infections.

Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.