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.

Stellate cells are in utero markers of pancreatic disease in cystic fibrosis.

BACKGROUND: Pancreatic fibrosis is an early diagnostic feature of the common inherited disorder cystic fibrosis (CF). Many people with CF (pwCF) are pancreatic insufficient from birth and the replacement of acinar tissue with cystic lesions and fibrosis is a progressive phenotype that may later lead to diabetes. Little is known about the initiating events in the fibrotic process though it may be a sequela of inflammation in the pancreatic ducts resulting from loss of CFTR impairing normal fluid secretion. Here we use a sheep model of CF (CFTR-/-) to examine the evolution of pancreatic disease through gestation. METHODS: Fetal pancreas was collected at six time points from 50-days of gestation through to term, which is equivalent to ~ 13 weeks to term in human. RNA was extracted from tissue for bulk RNA-seq and single cells were prepared from 80-day, 120-day and term samples for scRNA-seq. Data were validated by immunochemistry. RESULTS: Transcriptomic evidence from bulk RNA-seq showed alterations in the CFTR-/- pancreas by 65-days of gestation, which are accompanied by marked pathological changes by 80-days of gestation. These include a fibrotic response, confirmed by immunostaining for COL1A1, αSMA and SPARC, together with acinar loss. Moreover, using scRNA-seq we identify a unique cell population that is significantly overrepresented in the CFTR-/- animals at 80- and 120-days gestation, as are stellate cells at term. CONCLUSION: The transcriptomic changes and cellular imbalance that we observe likely have pivotal roles in the evolution of CF pancreatic disease and may provide therapeutic opportunities to delay or prevent pancreatic destruction in CF.

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.

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.

Comparative Histopathologic Lesions of the Male Reproductive Tract during Acute Infection of Zika Virus in AG129 and Ifnar-/- Mice.

An understanding of the pathogenesis of infection with the Zika virus in the male reproductive tract is vital for the development of vaccines and antivirals that will limit or prevent sexual transmission. Two common immunocompromised mouse strains used in transmission studies-male with genes encoding interferon types I and II receptor gene knockout (IFNAR/IFNGR; AG129) and with interferon type 1 receptor knockout (Ifnar-/-) were infected with a Puerto Rican Zika virus isolate (PRVABC59), and pathology was assessed 5 to 11 days after infection. Virus was detected by immunohistochemistry and quantitative RT-PCR in the testicle and epididymis of AG129 and Ifnar-/- mice, and by immunohistochemistry in the prostate and seminal vesicle of infected AG129 mice. Severe disease manifestations initiating as epididymitis and progressing to orchitis were observed in both models, with more severe inflammation noted in the AG129 mouse strain. Significant inflammation was not observed in any evaluated accessory sex gland at any point during infection. Time-course analysis of infection revealed an increase in the severity of disease within the epididymis of both strains, indicating a potential route of sexual transmission. Male mice with Ifnar-/- may better recapitulate Zika virus in humans and provide insight into the mechanism of sexual transmission, due to milder histopathologic lesions, the presence of histologically normal sperm in epididymal tubules, and an ability to survive the acute phase of disease.

Natural History and Pathogenesis of Wild-Type Marburg Virus Infection in STAT2 Knockout Hamsters.

Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.

Modeling Severe Fever with Thrombocytopenia Syndrome Virus Infection in Golden Syrian Hamsters: Importance of STAT2 in Preventing Disease and Effective Treatment with Favipiravir.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease endemic in parts of Asia. The etiologic agent, SFTS virus (SFTSV; family Bunyaviridae, genus Phlebovirus) has caused significant morbidity and mortality in China, South Korea, and Japan, with key features of disease being intense fever, thrombocytopenia, and leukopenia. Case fatality rates are estimated to be in the 30% range, and no antivirals or vaccines are approved for use for treatment and prevention of SFTS. There is evidence that in human cells, SFTSV sequesters STAT proteins in replication complexes, thereby inhibiting type I interferon signaling. Here, we demonstrate that hamsters devoid of functional STAT2 are highly susceptible to as few as 10 PFU of SFTSV, with animals generally succumbing within 5 to 6 days after subcutaneous challenge. The disease included marked thrombocytopenia and inflammatory disease characteristic of the condition in humans. Infectious virus titers were present in the blood and most tissues 3 days after virus challenge, and severe inflammatory lesions were found in the spleen and liver samples of SFTSV-infected hamsters. We also show that SFTSV infection in STAT2 knockout (KO) hamsters is responsive to favipiravir treatment, which protected all animals from lethal disease and reduced serum and tissue viral loads by 3 to 6 orders of magnitude. Taken together, our results provide additional insights into the pathogenesis of SFTSV infection and support the use of the newly described STAT2 KO hamster model for evaluation of promising antiviral therapies. IMPORTANCE: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease for which there are currently no therapeutic options or available vaccines. The causative agent, SFTS virus (SFTSV), is present in China, South Korea, and Japan, and infections requiring medical attention result in death in as many as 30% of the cases. Here, we describe a novel model of SFTS in hamsters genetically engineered to be deficient in a protein that helps protect humans and animals against viral infections. These hamsters were found to be susceptible to SFTSV and share disease features associated with the disease in humans. Importantly, we also show that SFTSV infection in hamsters can be effectively treated with a broad-spectrum antiviral drug approved for use in Japan. Our findings suggest that the new SFTS model will be an excellent resource to better understand SFTSV infection and disease as well as a valuable tool for evaluating promising antiviral drugs.

Congenital Short-Bowel Syndrome in an Adult Dog.

A 3.5-year-old, neutered male pit bull dog was euthanized following an approximately 1-year history of intractable diarrhea and weight loss of undetermined cause. At necropsy, the dog was emaciated. The ratio of total intestinal length (duodenum to rectum) to crown-to-rump length was 2.5, in contrast to an average of 5.3 (range, 3.7-6.1) in 10 control dogs examined at necropsy. There was diffuse dilation of the intestinal lumen, consistent with congenital intestinal hypoplasia resulting in short-bowel syndrome. Histologically, the intestinal mucosal was hyperplastic, further supporting the diagnosis of short-bowel syndrome. To the authors' knowledge, this is the first case of this condition in the veterinary literature.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-ß1.

INTRODUCTION: Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-ß1 and investigated the changes in the cardiac structure and function leading to AF. METHODS AND RESULTS: Transgenic goats with cardiac specific overexpression of constitutively active TGF-ß1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed. CONCLUSION: A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-ß1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility.

Phenotypic characterization of transgenic Japanese medaka (Oryzias latipes) that express a red fluorescent protein in hepatocytes.

Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.

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.

Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe arenavirus infection in AG129 mice.

BACKGROUND: Tacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV infection, but the pathogenic mechanisms contributing to the lethal disease are unclear. METHODS: To gain insights into the pathogenesis of TCRV infection in AG129 mice, we assessed hematologic and cytokine responses during the course of infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute infection, and measured serum and tissue viral titers out to 40 days post-infection to determine whether the virus is ultimately cleared in recovering mice. RESULTS: We found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2-3 weeks post-infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study. CONCLUSIONS: Our findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.

Anchoring hepatic gene expression with development of fibrosis and neoplasia in a toxicant-induced fish model of liver injury.

Fish have been used as laboratory models to study hepatic development and carcinogenesis but not for pathogenesis of hepatic fibrosis. In this study, a dimethylnitrosamine-induced fish model of hepatic injury was developed in Japanese medaka (Oryzias latipes) and gene expression was anchored with the development of hepatic fibrosis and neoplasia. Exposed livers exhibited mild hepatocellular degenerative changes 2 weeks' postexposure. Within 6 weeks, hepatic fibrosis/cirrhosis was evident with development of neoplasia by 10 weeks. Stellate cell activation and development of fibrosis was associated with upregulation of transforming growth factor beta 1 (tgfb1), tgfb receptor 2, mothers against decapentaplegic homolog 3 (smad3a), smad3b, beta-catenin (ctnnb1), myc, matrix metalloproteinase (mmp2), mmp14a, mmp14b, tissue inhibitors of metalloproteinase (timp) 2a, timp2b, timp3, collagen type I alpha 1a (col1a1a), and col1a1b and a less pronounced increase in mmp13 and col4a1 expression. Tgfb receptor I expression was unchanged. Immunohistochemistry suggested that biliary epithelial cells and stellate cells were the main producers of TGF-ß1. This study identified a group of candidate genes likely to be involved in the development of hepatic fibrosis and demonstrated that the TGF-ß pathway likely plays a major role in the pathogenesis. These results support the medaka as a viable fish model of hepatic fibrosis.

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.

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.

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.

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.

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.

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.