SARS-CoV-2 delta variant in African lions (Panthera leo) and humans at Utah's Hogle Zoo, USA, 2021-22.

AIMS: We conducted a One Health investigation to assess the source and transmission dynamics of SARS-CoV-2 infection in African lions (Panthera leo) at Utah's Hogle Zoo in Salt Lake City from October 2021 to February 2022. METHODS AND RESULTS: Following observation of respiratory illness in the lions, zoo staff collected pooled faecal samples and individual nasal swabs from four lions. All specimens tested positive for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR). The resulting investigation included: lion observation; RT-PCR testing of lion faeces every 1-7 days; RT-PCR testing of lion respiratory specimens every 2-3 weeks; staff interviews and RT-PCR testing; whole-genome sequencing of viruses from lions and staff; and comparison with existing SARS-CoV-2 human community surveillance sequences. In addition to all five lions, three staff displayed respiratory symptoms. All lions recovered and no hospitalizations or deaths were reported among staff. Three staff reported close contact with the lions in the 10 days before lion illness onset, one of whom developed symptoms and tested positive for SARS-CoV-2 on days 3 and 4, respectively, after lion illness onset. The other two did not report symptoms or test positive. Two staff who did not have close contact with the lions were symptomatic and tested positive on days 5 and 8, respectively, after lion illness onset. We detected SARS-CoV-2 RNA in lion faeces for 33 days and in lion respiratory specimens for 14 weeks after illness onset. The viruses from lions were genetically highly related to those from staff and two contemporaneous surveillance specimens from Salt Lake County; all were delta variants (AY.44). CONCLUSIONS: We did not determine the sources of these infections, although human-to-lion transmission likely occurred. The observed period of respiratory shedding was longer than in previously documented SARS-CoV-2 infections in large felids, indicating the need to further assess duration and potential implications of shedding.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022.

We describe the pathology of natural infection with highly pathogenic avian influenza A(H5N1) virus of Eurasian lineage Goose/Guangdong clade 2.3.4.4b in 67 wild terrestrial mammals throughout the United States during April 1‒July 21, 2022. Affected mammals include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals showed primarily neurologic signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung.

The precise molecular events initiating human lung disease are often poorly characterized. Investigating prenatal events that may underlie lung disease in later life is challenging in man, but insights from the well-characterized sheep model of lung development are valuable. Here, we determine the transcriptomic signature of lung development in wild-type sheep (WT) and use a sheep model of cystic fibrosis (CF) to characterize disease associated changes in gene expression through the pseudoglandular, canalicular, saccular, and alveolar stages of lung growth and differentiation. Using gene ontology process enrichment analysis of differentially expressed genes at each developmental time point, we define changes in biological processes (BP) in proximal and distal lung from WT or CF animals. We also compare divergent BP in WT and CF animals at each time point. Next, we establish the developmental profile of key genes encoding components of ion transport and innate immunity that are pivotal in CF lung disease and validate transcriptomic data by RT-qPCR. Consistent with the known pro-inflammatory phenotype of the CF lung after birth, we observe upregulation of inflammatory response processes in the CF sheep distal lung during the saccular stage of prenatal development. These data suggest early commencement of therapeutic regimens may be beneficial.

Basal Diet Fed to Recipient Mice Was the Driving Factor for Colitis and Colon Tumorigenesis, despite Fecal Microbiota Transfer from Mice with Severe or Mild Disease.

Consumption of the total Western diet (TWD) in mice has been shown to increase gut inflammation, promote colon tumorigenesis, and alter fecal microbiome composition when compared to mice fed a healthy diet, i.e., AIN93G (AIN). However, it is unclear whether the gut microbiome contributes directly to colitis-associated CRC in this model. The objective of this study was to determine whether dynamic fecal microbiota transfer (FMT) from donor mice fed either the AIN basal diet or the TWD would alter colitis symptoms or colitis-associated CRC in recipient mice, which were fed either the AIN diet or the TWD, using a 2 × 2 factorial experiment design. Time-matched FMT from the donor mice fed the TWD did not significantly enhance symptoms of colitis, colon epithelial inflammation, mucosal injury, or colon tumor burden in the recipient mice fed the AIN diet. Conversely, FMT from the AIN-fed donors did not impart a protective effect on the recipient mice fed the TWD. Likewise, the composition of fecal microbiomes of the recipient mice was also affected to a much greater extent by the diet they consumed than by the source of FMT. In summary, FMT from the donor mice fed either basal diet with differing colitis or tumor outcomes did not shift colitis symptoms or colon tumorigenesis in the recipient mice, regardless of the basal diet they consumed. These observations suggest that the gut microbiome may not contribute directly to the development of disease in this animal model.

Human IVIG treatment in a neurological disease model for Enterovirus A71 infection in 28-day-old AG129 mice.

Enterovirus A71 can cause serious neurological disease in young children. Animal models for EV-A71 are needed to evaluate potential antiviral therapies. Existing models have limitations, including lack of lethality or crucial disease signs. Here we report the development of an EV-A71 model in 28-day-old mice. Virus was serially passaged until it produced consistent lethality and rear-limb paralysis. Onset of disease occurred between days 6-9 post-infection, with mortality following weight loss and neurological signs on days 9-14. In addition, a single administration of human intravenous immunoglobulin at doses of 200, 400 and 800 mg/kg at 4h post-infection was evaluated in the model. Protection from weight loss, neurological signs, and mortality (between 50 and 89%) were observed at doses of 400 mg/kg or greater. Based on these results, IVIG was selected for use as a positive control in this acute model, and suggest that IVIG is a potential therapeutic for EV-A71 infections.

Disease Surveillance of Cougars (Puma concolor) in Utah, USA.

Adult, free-ranging cougars (Puma concolor) were sampled in three regions of Utah, US, from 2018 to 2021. A total of 68% (23/34) of the sampled cougars had antibodies to feline parvovirus, 15% (5/33) to canine distemper virus, 18% (6/34) to calicivirus, and 22% (8/37) to Yersinia pestis. Forty-one percent (13/32) had IgG antibodies to Toxoplasma gondii and 6% (2/33) to feline immunodeficiency virus, and 3% (1/32) were positive for Dirofilaria immitis (heartworm) antigen. All were seronegative for Toxoplasma gondii IgM, feline enteric coronavirus, SARS-CoV-2, feline leukemia virus, feline herpesvirus, and Francisella tularensis. Tapeworms and Toxascaris leonina eggs were detected in the feces. The disease exposures detected were similar to what has been reported from cougar populations in other western US states, and the current level of exposures is unlikely to have a negative impact on the state's population.

Early developmental phenotypes in the cystic fibrosis sheep model.

Highly effective modulator therapies for cystic fibrosis (CF) make it a treatable condition for many people. However, although CF respiratory illness occurs after birth, other organ systems particularly in the digestive tract are damaged before birth. We use an ovine model of CF to investigate the in utero origins of CF disease since the sheep closely mirrors critical aspects of human development. Wildtype (WT) and CFTR -/- sheep tissues were collected at 50, 65, 80, 100, and 120 days of gestation and term (147 days) and used for histological, electrophysiological, and molecular analysis. Histological abnormalities are evident in CFTR-/- -/-  animals by 80 days of gestation, equivalent to 21 weeks in humans. Acinar and ductal dilation, mucus obstruction, and fibrosis are observed in the pancreas; biliary fibrosis, cholestasis, and gallbladder hypoplasia in the liver; and intestinal meconium obstruction, as seen at birth in all large animal models of CF. Concurrently, cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short circuit current is present in WT tracheal epithelium by 80 days gestation and is absent from CFTR -/- tissues. Transcriptomic profiles of tracheal tissues confirm the early expression of CFTR and suggest that its loss does not globally impair tracheal differentiation.

Development and characterization of type I interferon receptor knockout sheep: A model for viral immunology and reproductive signaling.

Type I interferons (IFNs) initiate immune responses to viral infections. Their effects are mediated by the type I IFN receptor, IFNAR, comprised of two subunits: IFNAR1 and IFNAR2. One or both chains of the sheep IFNAR were disrupted in fetal fibroblast lines using CRISPR/Cas9 and 12 lambs were produced by somatic cell nuclear transfer (SCNT). Quantitative reverse transcription-polymerase chain reaction for IFN-stimulated gene expression showed that IFNAR deficient sheep fail to respond to IFN-alpha. Furthermore, fibroblast cells from an IFNAR2 -/- fetus supported significantly higher levels of Zika virus (ZIKV) replication than wild-type fetal fibroblast cells. Although many lambs have died from SCNT related problems or infections, one fertile IFNAR2 -/- ram lived to over 4 years of age, remained healthy, and produced more than 80 offspring. Interestingly, ZIKV infection studies failed to demonstrate a high level of susceptibility. Presumably, these sheep compensated for a lack of type I IFN signaling using the type II, IFN-gamma and type III, IFN-lambda pathways. These sheep constitute a unique model for studying the pathogenesis of viral infection. Historical data supports the concept that ruminants utilize a novel type I IFN, IFN-tau, for pregnancy recognition. Consequently, IFNAR deficient ewes are likely to be infertile, making IFNAR knockout sheep a valuable model for studying pregnancy recognition. A breeding herd of 32 IFNAR2 +/- ewes, which are fertile, has been developed for production of IFNAR2 -/- sheep for both infection and reproduction studies.

Health Screening of American Beavers (Castor canadensis) in Utah, USA.

American beavers (Castor canadensis), trapped between 2017 and 2020 for the purpose of translocation, underwent hematologic, serologic, and fecal examinations. Eight of 73 beavers were seropositive for Leptospira spp. and 1/49 seropositive for Toxoplasma gondii. Two of 40 beavers tested positive for Giardia spp., and 22/44 beavers had strongyle-type ova in the feces; one was positive for coccidia. Hematologic variables were largely within published reference ranges for captive beavers. Within the specific ecosystems sampled, the disease prevalence in beavers appeared low; however, prophylactic deworming is recommended prior to translocation. Further, appropriate personal protective equipment should be used when handling beavers, to prevent zoonotic infection with Giardia spp.

Trophoblast emboli in the lung of a snowshoe hare (Lepus americanus).

Spontaneous migration of placental trophoblasts into maternal blood vessels and embolization to other organs (ie, lung, adrenal gland, spleen, and liver) occurs in women and certain animals with hemochorial placentation. Although considered incidental in most species, increased incidence and numbers of trophoblast emboli are reported in women with gestational diseases with arterial hypertension (pre-eclampsia and eclampsia). To the best of our knowledge, trophoblast emboli have not been reported in lagomorphs. This case report describes the identification of trophoblast emboli in the lung of a wild snowshoe hare (Lepus americanus). Death of this hare was attributed to pulmonary hemorrhages and hemothorax, but a definitive cause for the hemorrhages was not determined. It is unclear whether trophoblast embolism normally occurs in this species and represents an incidental finding, or whether it possibly contributed to rupture of pulmonary or thoracic blood vessels leading to hemorrhage.

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys.

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

Pandemic lineage 2009 H1N1 influenza A virus infection in farmed mink in Utah.

Mink are susceptible to infection with influenza A virus (IAV) of swine and human origin. In 2019, a Utah mink farm had an outbreak of respiratory disease in kits caused by infection with the pandemic influenza A(H1N1)2009 virus [A(H1N1)pdm09]. In 3 wk, ~325, 1-2-wk-old kits died (10% mortality in kits). All deaths occurred in a single barn that housed 640 breeding females. No clinical signs or deaths occurred among adult mink. Five dead kits and 3 euthanized female mink were autopsied. All kits had moderate-to-severe neutrophilic and lymphohistiocytic interstitial pneumonia; adult mink had minimal-to-moderate lymphohistiocytic bronchointerstitial pneumonia. Immunohistochemistry and real-time PCR targeting the matrix gene detected IAV in lung of kits and adults. Virus isolation and genetic analysis identified the A(H1N1)pdm09 virus. The source of the virus was not determined but is thought to be the result of reverse zoonosis. Our case emphasizes the need for close monitoring on mink farms for interspecies transmission of IAV and for safe work practices on farms and in diagnostic laboratories. Additionally, a pandemic virus may continue to circulate at low levels long after the global event is declared over.

Investigation into Causes of Antler Deformities in Mule Deer (Odocoileus hemionus) Bucks in Southern Utah, USA.

We investigated causes of antler deformities in mule deer (Odocoileus hemionus) bucks from the Paunsaugunt Plateau in southern Utah, US. A total of 10 hunter-harvested and nine live-captured bucks with antler deformities and six hunter-harvested and 43 live-captured bucks with normal antlers were included in the study. All were screened by serology for exposure to epizootic hemorrhagic disease virus (EHDV) types 1, 2, and 6, bluetongue virus, Brucella abortus, Brucella ovis, caprine arthritis, encephalitis virus, and bovine viral diarrhea virus (BVDV) types 1 and 2. Serum testosterone and trace minerals concentrations were measured, and whole-blood counts evaluated. Testicular tissue from the hunter-harvested bucks was tested by quantitative PCR for EHDV-1, -2, and -6. All bucks with antler deformities had low to non-detectable serum testosterone concentrations from end-stage fibrosing orchitis, and EHDV-2 was detected by quantitative PCR in one of the testicular tissues tested. All bucks with antler deformities were seropositive for EHDV-2. In comparison, only 53% of bucks with normal antlers were EHDV-2 seropositive. More than 67% of hunter-harvested and live-captured bucks had antibodies to BVDV-1 and BVDV-2, probably because of high cattle exposure in the area. Our results support previous research linking infection with EHDV-2 to development of antler deformities; however, it remains unclear why some infected bucks develop testicular fibrosis, and others recover from the infection.

Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR -/- sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder.

The Western Dietary Pattern Combined with Vancomycin-Mediated Changes to the Gut Microbiome Exacerbates Colitis Severity and Colon Tumorigenesis.

Previous work by our group using a mouse model of inflammation-associated colorectal cancer (CAC) showed that the total Western diet (TWD) promoted colon tumor development. Others have also shown that vancomycin-mediated changes to the gut microbiome increased colorectal cancer (CRC). Therefore, the objective of this study was to determine the impact of vancomycin on colon tumorigenesis in the context of a standard mouse diet or the TWD. A 2 × 2 factorial design was used, in which C57Bl/6J mice were fed either the standard AIN93G diet or TWD and with vancomycin in the drinking water or not. While both the TWD and vancomycin treatments independently increased parameters associated with gut inflammation and tumorigenesis compared to AIN93G and plain water controls, mice fed the TWD and treated with vancomycin had significantly increased tumor multiplicity and burden relative to all other treatments. Vancomycin treatment significantly decreased alpha diversity and changed the abundance of several taxa at the phylum, family, and genus levels. Conversely, basal diet had relatively minor effects on the gut microbiome composition. These results support our previous research that the TWD promotes colon tumorigenesis and suggest that vancomycin-induced changes to the gut microbiome are associated with higher tumor rates.

Type I interferon underlies severe disease associated with Junín virus infection in mice.

Junín virus (JUNV) is one of five New World mammarenaviruses (NWMs) that causes fatal hemorrhagic disease in humans and is the etiological agent of Argentine hemorrhagic fever (AHF). The pathogenesis underlying AHF is poorly understood; however, a prolonged, elevated interferon-α (IFN-α) response is associated with a negative disease outcome. A feature of all NWMs that cause viral hemorrhagic fever is the use of human transferrin receptor 1 (hTfR1) for cellular entry. Here, we show that mice expressing hTfR1 develop a lethal disease course marked by an increase in serum IFN-α concentration when challenged with JUNV. Further, we provide evidence that the type I IFN response is central to the development of severe JUNV disease in hTfR1 mice. Our findings identify hTfR1-mediated entry and the type I IFN response as key factors in the pathogenesis of JUNV infection in mice.

Vascular Leak and Hypercytokinemia Associated with Severe Fever with Thrombocytopenia Syndrome Virus Infection in Mice.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever (VHF) endemic to China, South Korea, Japan, and Vietnam. Here we characterize the pathogenesis and natural history of disease in IFNAR-/- mice challenged with the HB29 strain of SFTS virus (SFTSV) and demonstrate hallmark features of VHF such as vascular leak and high concentrations of proinflammatory cytokines in blood and tissues. Treatment with FX06, a natural plasmin digest product of fibrin in clinical development as a treatment for vascular leak, reduced vascular permeability associated with SFTSV infection but did not significantly improve survival outcome. Further studies are needed to assess the role of vascular compromise in the SFTS disease process modeled in IFNAR-/- mice.

Zika Virus Associated Pathology and Antigen Presence in the Testicle in the Absence of Sexual Transmission During Subacute to Chronic Infection in a Mouse Model.

Zika virus (ZIKV) is an arboviral infection that has been shown to be sexually transmitted. The study outlined herein aims to determine if accessory sex glands and epididymal epithelial cells are sources of viral persistence in subacute and chronic ZIKV infection, and if infection of these organs is important in sexual transmission during long-term (chronic) infection. Male interferon type I receptor knockout (Ifnar-/-) mice were challenged with ZIKV and reproductive tissues were harvested 14 and 35 days post infection (DPI) for inoculation studies and 14, 35 and 70 DPI for histopathology. Artificial insemination fluid derived from epididymal flush and seminal plasma from the prostate and seminal vesicle was obtained from ZIKV inoculated and sham-infected males. Naïve interferon type I and II receptor knockout (AG129) female mice were pre-treated with progesterone and inoculated intravaginally with artificial insemination fluid from ZIKV-infected males. ZIKV RNA was detected in the artificial insemination fluid generated from epididymal flush or seminal plasma from ZIKV infected males at 14 and 35 DPI. ZIKV antigens were only detected in seminiferous tubules at 14 DPI. Epididymal epithelial cells did not show ZIKV antigen immunoreactivity at 14, 35 or 70 DPI. Severe fibrosing orchitis (end stage orchitis) was observed at 35 and 70 DPI. Mild inflammation and peri-tubular fibrosis were observed in the epididymis following clearance of virus. Viral RNA was not detected by PCR in whole blood samples of females from any intravaginal experimental group and only detected in 20% of subcutaneously inoculated animals (derived from 1 experimentally infected male) at 35 DPI. While ZIKV RNA and antigens can be detected in the male reproductive tract at 14 DPI and RNA can also be detected at 35 DPI, intravaginal inoculation of artificial insemination fluid from these time-points failed to result in viremia in naïve females inoculated intravaginally. These studies support the hypothesis that epididymal epithelial cells are critical to sexual transmission in immunocompromised mice. Additionally, acute but not chronic male reproductive tract infection with ZIKV results in infectious virus capable of being sexually transmitted in mice.

Incomplete Urethral Duplication Associated with a Dermoid Cyst within a Vascular Hamartoma in a Female Dog.

A seven-year-old spayed female Labrador retriever presented for necropsy following an acute history of thrombocytopenia, anemia, leukocytosis and abdominal effusion. A 2 × 3 × 10 cm, cylindrical to tubular, mottled red-to-tan mass extended from the caudal pelvic cavity caudally and ventrally under the dermis along the caudal aspect of the left pelvic limb adjacent to the semimembranosus and semitendinosus musculature. Histologic examination of the mass revealed a singular central lumen lined by urothelium that multifocally transitioned into non-keratinizing, stratified squamous epithelium associated with few hair follicles and sweat glands. The lumen was surrounded by a dense collagenous stroma containing numerous, variably sized blood vessels. The combination of lesions was consistent with a diagnosis of incomplete urethral duplication associated with a dermoid cyst and vascular hamartoma. To the authors' knowledge, this is the first report of an incomplete urethral duplication associated with a dermoid cyst within a vascular hamartoma.