First reported case in an Irish flock of MCF- like systemic necrotizing vasculitis in sheep associated with ovine herpesvirus 2.

BACKGROUND: Ovine gammaherpesvirus 2 (OvHV-2) is the causative agent of sheep associated malignant catarrhal fever (MCF). As sheep are the adapted host for OvHV-2, it is generally presumed that infection is not associated with disease in this species. However, a recent case review combined in-situ hybridisation, PCR and histopathology and correlated the viral distribution with systemic necrotizing vasculitis and concluded OvHV-2 was the likely agent responsible for sporadic, MCF-like vascular disease in sheep. CASE PRESENTATION: Using similar methods this case study reports on the findings of the first reported cases in an Irish Flock of MCF- like systemic necrotizing vasculitis in sheep associated with OvHV-2. Sheep A, a 16-month-old Texel-cross hogget displayed signs of ill- thrift, Sheep B, a nine-month-old Belclare-cross lamb, was found dead having displayed no obvious symptoms. Both cases occurred on the same farm, however the animals were not related. Lymphohistiocytic vasculitis of various tissues was the predominant histopathological finding in both animals. CONCLUSION: By combining histopathology, PCR and in-situ hybridisation results, MCF- like systemic necrotizing vasculitis associated with OvHV-2 has been diagnosed for the first time in an Irish flock.

Reply to Piewbang et al. Domestic Cat Hepadnavirus Antigens in Lymphoma Tissues. Comment on "Beatty et al. Domestic Cat Hepadnavirus and Lymphoma. Viruses 2023, 15, 2294".

We are grateful to the authors for providing additional data to demonstrate the presence of domestic cat hepadnavirus in lymphoma tissues [...].

Amdoparvovirus-associated disease in red pandas (Ailurus fulgens).

The roster of amdoparvoviruses (APVs) in small carnivores is growing rapidly, but in most cases, the consequences of infection are poorly understood. Red panda amdoparvovirus (RPAV) is highly prevalent in zoo-housed red pandas and has been detected in both healthy and sick animals. Clarifying the clinical impact of RPAV in this endangered species is critical, and zoological collections offer a unique opportunity to examine viral disease association in carefully managed populations. We evaluated the potential impact of RPAV in captive red pandas with a combination of prospective and retrospective analyses. First, we collected feces from 2 healthy animals from one collection over a 6-year period and detected virus in 72/75 total samples, suggesting that RPAV can be a long-term subclinical infection. We next investigated the infections using a retrospective study of infection status and tissue distribution in a cohort of necropsied animals. We performed polymerase chain reaction and in situ hybridization on 43 necropsy cases from 4 zoo collections (3 from the United States, 1 from Europe, 1997-2022). RPAV was present in these populations for at least 2 decades before its discovery and is detectable in common and significant lesions of zoo-housed red pandas, including myocarditis (3/3 cases), nephritis (9/10), and interstitial pneumonia (2/4). RPAV is also detectable in sporadic lesions, including multisystemic pyogranulomatous inflammation, oral/pharyngeal mucosal inflammation, and dermatitis. The colocalization of virus with lesions supports a role in causation, suggesting that despite the apparently persistent and subclinical carriage of most infections, RPAV may have a significant impact in zoo collections.

Domestic Cat Hepadnavirus and Lymphoma.

We write to comment on Piewbang C et al [...].

Amdoparvovirus-associated disease in striped skunks (Mephitis mephitis).

Disease caused by the archetypical amdoparvovirus (APV), Aleutian mink disease virus (AMDV), has been well studied, but APV infections in other carnivores are poorly understood. Skunk amdoparvovirus (SKAV), one of a handful of newly discovered APVs, is apparently species-specific in striped skunks (Mephitis mephitis) and has a high prevalence across North America. We have evaluated the infection status and viral tissue distribution in a cohort of 26 free-ranging California skunks from a single rehabilitation facility who were euthanized due to poor prognosis for recovery from neurologic disease. SKAV was detected in the majority of this cohort, and virus was associated with a spectrum of lesions including tubulointerstitial nephritis, meningoencephalitis, myocarditis, and arteritis. Affected tissue and patterns of inflammation were partially overlapping with those of AMDV infection but were notably distinct in the kidney.

Severe Acute Respiratory Syndrome Coronavirus 2 Vasculopathy in a Syrian Golden Hamster Model.

Clinical evidence of vascular dysfunction and hypercoagulability as well as pulmonary vascular damage and microthrombosis are frequently reported in severe cases of human coronavirus disease 2019 (COVID-19). Syrian golden hamsters recapitulate histopathologic pulmonary vascular lesions reported in patients with COVID-19. Herein, special staining techniques and transmission electron microscopy further define vascular pathologies in a Syrian golden hamster model of human COVID-19. The results show that regions of active pulmonary inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are characterized by ultrastructural evidence of endothelial damage with platelet marginalization and both perivascular and subendothelial macrophage infiltration. SARS-CoV-2 antigen/RNA was not detectable within affected blood vessels. Taken together, these findings suggest that the prominent microscopic vascular lesions in SARS-CoV-2-inoculated hamsters likely occur due to endothelial damage followed by platelet and macrophage infiltration.

Longevity and mortality in cats: A single institution necropsy study of 3108 cases (1989-2019).

Client-owned cats who underwent a post-mortem examination (n = 3,108) at a veterinary medical teaching hospital between 1989 and 2019 were studied to determine longevity and factors affecting mortality. Demographic factors, environmental factors, age, and causes of death were assessed. Sexes included 5.66% intact females, 39.86% spayed females, 6.95% intact males and 47.49% neutered males. 84.2% were mixed breed cats. Age at death was known for 2,974 cases with a median of 9.07 years. Cancer was the most common pathophysiologic cause of death (35.81%) and was identified in 41.3% of cats. When categorized by organ system, mortality was most attributed to multiorgan/systemic (21.72%). Renal histologic abnormalities were noted in 62.84% of cats but was considered the primary cause of death in only 13.06% of cats. Intact female and male cats had significantly shorter lifespans than their spayed or neutered counterparts. FeLV positive status was associated with decreased longevity (P<0.0001) while FIV status was not. This study reports on risk factors associated with mortality and highlights areas of research that may contribute to improved lifespan in cats.

PULMONARY AND COELOMIC MYCOSES DUE TO METARHIZIUM AND BEAUVERIA SPECIES IN REPTILES.

This report documents cases of fatal pulmonary mycosis caused by entomopathogenic fungi in the genera Metarhizium and Beauveria (Order Hypocreales) in a loggerhead sea turtle (Caretta caretta), a Chinese alligator (Alligator sinensis), two gopher tortoises (Gopherus polyphemus), a Cuvier's dwarf caiman (Paleosuchus palpebrosus), a false gharial (Tomistoma schlegelii), a green sea turtle (Chelonia mydas), and a Kemp's ridley sea turtle (Lepidochelys kempii), and a case of granulomatous coelomitis in a hawksbill sea turtle (Eretmochelys imbricata). Fungi identified in these cases included Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Metarhizium robertsii, and one case of infection by a novel Metarhizium species. The animals were either housed at zoos or brought into rehabilitation from the wild. Although the majority of animals had comorbidities, the fungal infections were believed to be the primary cause of death. Fungal susceptibility testing was performed on two Beauveria spp. isolates, and revealed lower minimum inhibitory concentrations for itraconazole and voriconazole when compared to terbinafine and fluconazole. This case series demonstrates that a variety of reptile species from different orders are vulnerable to infection with Metarhizium, and multiple species of sea turtle are susceptible to infection with Beauveria.

Hepadnavirus DNA Is Detected in Canine Blood Samples in Hong Kong but Not in Liver Biopsies of Chronic Hepatitis or Hepatocellular Carcinoma.

Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.

Zika virus persistence in the male macaque reproductive tract.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

Systemic proliferative arteriopathy and hypophysitis in a cow with chronic ovine herpesvirus 2-induced malignant catarrhal fever.

Malignant catarrhal fever (MCF) is a severe, systemic, lymphoproliferative disease affecting domestic ruminants, caused by a group of MCF viruses in the genus Macavirus. Infection of cattle and bison with ovine herpesvirus 2 (OvHV2) is economically significant in North America. Sheep are the reservoir host of the virus, and only rarely manifest disease. Cattle and bison, however, frequently have lymphoproliferation, mucosal ulceration, and systemic vasculitis. OvHV2-induced MCF in cattle and bison is often fatal, with clinical recovery reported only rarely. Chronic cases are uncommon, but vascular changes of variable severity and ocular lesions have been described. Here we present a case of chronic MCF in a cow with proliferative arteriopathy, systemic vasculitis, and OvHV2-associated hypophysitis. We demonstrated OvHV2 nucleic acid in affected tissues with in situ hybridization.

Extracellular Vesicles (EVs) Are Copurified with Feline Calicivirus, yet EV-Enriched Fractions Remain Infectious.

Feline calicivirus (FCV) is a major cause of upper respiratory disease in cats and is often used as a model for human norovirus, making it of great veterinary and human medical importance. However, questions remain regarding the route of entry of FCV in vivo. Increasing work has shown that extracellular vesicles (EVs) can be active in viral infectivity, yet there is no work examining the role of EVs in FCV infection. Here, we begin to address this knowledge gap by characterizing EVs produced by a feline mammary epithelial cell line (FMEC). We have confirmed that EVs are produced by infected and mock-infected FMECs and that both virions and EVs are coisolated with standard methods of virus purification. We also show that they can be enriched differentially by continuous iodixanol density gradient. EVs were enriched at a density of 1.10 g/mL confirmed by tetraspanin expression, size profile, and transmission electron microscopy (TEM). Maximum enrichment of FCV at a density of 1.18 g/mL was confirmed by titration, quantitative reverse transcriptase PCR (q-RT PCR), and TEM. However, infectious virus was recovered from nearly all samples. When used to infect in vitro epithelium, both EV-rich and virus-rich fractions had the same levels of infectiousness as determined by percentage of wells infected or titer achieved postinfection. These findings highlight the importance of coisolates during viral purification, showing that EVs may represent a parallel route of entry that has previously been overlooked. Additional experiments are necessary to explore the role of EVs in FCV infection. IMPORTANCE Feline calicivirus (FCV) is a common cause of upper respiratory infection in cats. Both healthy and infected cells produce small particles called extracellular vesicles (EVs), which are nanoparticles that act as messengers between cells and can be hijacked during viral infection. Historically, the role of EVs in viral infection has been overlooked, and subsequently no group has studied the role of EVs in FCV infection. We hypothesized that EVs may play a role in FCV infection. Here, we show that EVs are copurified with FCV when collecting virus. To study their individual effects, we successfully enrich for viral particles and EVs separately by taking advantage of their different densities. Our initial studies show that EV-enriched versus virus-enriched fractions are equally able to infect cells in culture. These findings highlight the need to both consider the purity of virus after purification and to further study EVs' role in natural FCV infection.

HYPERLIPIDEMIA AND XANTHOMATOSIS IN YELLOW-FOOTED ROCK WALLABIES (PETROGALE XANTHOPUS) UNDER MANAGED CARE.

Xanthomas are localized lipid deposits in organs with associated granulomatous inflammation. Xanthomatosis is a rare condition in both human and veterinary medicine and is often linked to inherited or acquired dyslipidemias. Three female yellow-footed rock wallabies (Petrogale xanthopus) at a single institution were diagnosed via biopsy with cutaneous xanthomas secondary to hypertriglyceridemia and hypercholesterolemia, and an additional two female yellow-footed rock wallabies were diagnosed with xanthomas at a second institution. All cases presented with cutaneous masses at the haired skin and paw pad junctions of the extremities, and/or mucocutaneous junctions of the face or urogenital tract. The clinically affected individuals were overconditioned or obese, had lipemic serum, and had elevations in blood cholesterol and triglyceride levels. When full lipid panels were performed, inverse high- and low-density lipoprotein fractions were observed. Six other individuals at the first institution had identical husbandry but were of more appropriate body condition, were normolipidemic, and had no xanthomas. One of the affected animals was also concurrently diagnosed with hepatic lipidosis via liver biopsy. Pedigree review and evaluation for underlying endocrine diseases such as hypothyroidism were performed. Because all affected animals were found to be related, a genetic predisposition is possible but requires further investigation. Consideration for the predisposition of some individuals for obesity, hyperlipidemia, and subsequent xanthoma formation should be factored in the husbandry and medical management of this species.

Distribution of canine distemper virus and nectin-4 in raccoon (Procyon lotor) skin.

Raccoons (Procyon lotor) are abundant in urban/wildland interfaces and are key sources of canine distemper virus (CDV) outbreaks in domestic, zoo, and free-ranging wildlife species. CDV is pantropic, which provides multiple potential routes of transmission (urine, respiratory secretions, feces), but the specific role of skin as a target of infection, as a diagnostic sample, or as a potential source of environmental persistence and transmission is unknown. We have characterized the distribution of CDV and its known receptor, nectin-4, in skin samples of 36 raccoons. Even with skin samples that were grossly and histologically normal, immunohistochemistry of skin was useful in the diagnosis of CDV infection, which was found in both epithelium and endothelium. Nectin-4 was codistributed with cellular targets of viral infection. Skin secretions, shed keratinocytes, and hair of CDV infected raccoons are all potential environmental fomites.

Natural disease and evolution of an Amdoparvovirus endemic in striped skunks (Mephitis mephitis).

Striped skunks (Mephitis mephitis) densely populate the human-animal interface of suburbia throughout North America. Skunks share that habitat with numerous related mesocarnivores, where increased contact, competition for shared food and water sources and other stressors contribute to increased exposure and susceptibility to viral infection. The recently identified skunk amdoparvovirus (SKAV) has been detected at high prevalence in skunks and occasionally in mink, but its distribution in North America is unknown. To understand the impact of SKAV in striped skunks and the risk posed to related species, we investigated the geographic distribution of SKAV, analysed its genetic diversity and evolutionary dynamics and evaluated viral distribution in tissues of infected animals to identify possible mechanisms of transmission. SKAV was detected in 72.5% (37/51) skunks and was present at high rates at all locations tested across North America. Analysis of the complete genomic sequence of 29 strains showed a clear geographic segregation, frequent recombination and marked differences in the evolutionary dynamics of the major structural (VP2) and non-structural (NS1) proteins. NS1 was characterized by a higher variability and a higher percentage of positively selected codons. This could indicate that antibody-mediated enhancement of infection occurs in SKAV, an infection strategy that may be conserved across amdoparvoviruses. Finally, in situ hybridization revealed virus in epithelium of the gastrointestinal tract, urinary tract and skin, indicating that viral transmission could occur via oronasal, faecal and/or urinary secretions, as well as from skin and hair. The endemicity of SKAV over large geographic distances and its high genetic diversity suggest a long-term virus-host association. Persistent shedding and high environmental stability likely contribute to efficient viral spread, simultaneously offering opportunities for cross-species transmission with consequent risk to sympatric species, including domestic animals and wildlife.

AMDOPARVOVIRUS INFECTIONS ARE PREVALENT, PERSISTENT, AND GENETICALLY DIVERSE IN ZOO-HOUSED RED PANDAS (AILURUS FULGENS).

Red pandas (Ailurus fulgens) are a globally endangered small carnivoran species and subjects of a robust ex situ conservation effort that includes animals housed in zoos. In 2018, red panda amdoparvovirus (RPAV) was discovered by metagenomics analyses of tissues from two geriatric red pandas, and in one case it was associated with significant lesions. Because RPAV was discovered in a single zoo cohort, it was unclear whether these infections represented a widely distributed, enzootic virus of red pandas or a localized 'spillover' from a different host species into this collection. The first goal of this study was to estimate the prevalence of RPAV in US zoos. The authors amplified RPAV from feces of 104 individual red pandas from 37 US zoos, and the virus was detected in 52/104 samples (50.0%). Next, to establish persistence of infection in individual animals, the authors tested serial samples in a single cohort over a 4.5-yr period, and virus was consistently shed by infected animals throughout the sampling period. Finally, full viral coding sequences were amplified and sequenced from three cases, and partial sequences of both the nonstructural and capsid genes were obtained for an additional 19 cases. RPAV is a genetically diverse but monophyletic viral species, and multiple viral lineages are present in US zoo-housed red pandas. The authors do not know how red pandas were originally infected, but RPAV is very common in red pandas in the United States, and infections are persistent-presumably for the lifetime of the animal.

Oropharyngeal Shedding of Gammaherpesvirus DNA by Cats, and Natural Infection of Salivary Epithelium.

Felis catus gammaherpesvirus-1 (FcaGHV1), a novel candidate oncogenic virus, infects cats worldwide. Whether the oropharynx is a site of virus shedding and persistence, and whether oronasal carcinomas harbor FcaGHV1 nucleic acid were investigated. In a prospective molecular epidemiological study, FcaGHV1 DNA was detected by cPCR in oropharyngeal swabs from 26/155 (16.8%) of cats. Oropharyngeal shedding was less frequently detected in kittens ≤3 months of age (5/94, 5.3%) than in older animals; >3 months to ≤1 year: 8/26, 30.8%, (p = 0.001, OR 7.91, 95% CI (2.320, 26.979)); >1 year to ≤6 years: 10/20, 50%, (p < 0.001, OR 17.8 95% CI (5.065, 62.557)); >6 years: 3/15, 33% (p = 0.078). Provenance (shelter-owned/privately owned) was not associated with shedding. In situ hybridization (ISH) identified FcaGHV1-infected cells in salivary glandular epithelium but not in other oronasal tissues from two of three cats shedding viral DNA in the oropharynx. In a retrospective dataset of 11 oronasopharyngeal carcinomas, a single tumor tested positive for FcaGHV1 DNA by ISH, a papillary carcinoma, where scattered neoplastic cells showed discrete nuclear hybridization. These data support the oronasopharynx as a site of FcaGHV1 shedding, particularly after maternal antibodies are expected to decline. The salivary epithelium is identified as a potential site of FcaGHV1 persistence. No evidence supporting a role for FcaGHV1 in feline oronasal carcinomas was found in the examined tumours.

Neuroanatomical abnormalities in a nonhuman primate model of congenital Zika virus infection.

We evaluated neuropathological consequences of fetal ZIKV exposure in rhesus monkeys, a translatable animal model for human neural development, by carrying out quantitative neuroanatomical analyses of the nearly full-term brains of fetuses infected with ZIKV and procedure-matched controls. For each animal, a complete cerebral hemisphere was evaluated using immunohistochemical (IHC) and neuroanatomical techniques to detect virus, identify affected cell types, and evaluate gross neuroanatomical abnormalities. IHC staining revealed the presence of ZIKV in the frontal lobe, which contained activated microglia and showed increased apoptosis of immature neurons. ZIKV-infected animals exhibited macrostructural changes within the visual pathway. Regional differences tracked with the developmental timing of the brain, suggesting inflammatory processes related to viral infiltration swept through the cortex, followed by a wave of cell death resulting in morphological changes. These findings may help explain why some infants born with normal sized heads during the ZIKV epidemic manifest developmental challenges as they age.

Feline Calicivirus Virulent Systemic Disease: Clinical Epidemiology, Analysis of Viral Isolates and In Vitro Efficacy of Novel Antivirals in Australian Outbreaks.

Feline calicivirus (FCV) causes upper respiratory tract disease (URTD) and sporadic outbreaks of virulent systemic disease (FCV-VSD). The basis for the increased pathogenicity of FCV-VSD viruses is incompletely understood, and antivirals for FCV-VSD have yet to be developed. We investigated the clinicoepidemiology and viral features of three FCV-VSD outbreaks in Australia and evaluated the in vitro efficacy of nitazoxanide (NTZ), 2'-C-methylcytidine (2CMC) and NITD-008 against FCV-VSD viruses. Overall mortality among 23 cases of FCV-VSD was 39%. Metagenomic sequencing identified five genetically distinct FCV lineages within the three outbreaks, all seemingly evolving in situ in Australia. Notably, no mutations that clearly distinguished FCV-URTD from FCV-VSD phenotypes were identified. One FCV-URTD strain likely originated from a recombination event. Analysis of seven amino-acid residues from the hypervariable E region of the capsid in the cultured viruses did not support the contention that properties of these residues can reliably differentiate between the two pathotypes. On plaque reduction assays, dose-response inhibition of FCV-VSD was obtained with all antivirals at low micromolar concentrations; NTZ EC50, 0.4-0.6 µM, TI = 21; 2CMC EC50, 2.7-5.3 µM, TI > 18; NITD-008, 0.5 to 0.9 µM, TI > 111. Investigation of these antivirals for the treatment of FCV-VSD is warranted.