Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

Detection of SARS-CoV-2 in a dog with hemorrhagic diarrhea.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. CASE PRESENTATION: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog's acute hemorrhagic diarrhea. CONCLUSION: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog's pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.

Genetic heterogeneity of dolphin morbilliviruses detected in the Spanish Mediterranean in inter-epizootic period.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the three dolphin morbillivirus (DMV) epizootics of 1990-1992, 2006-2008 and 2011 that affected Mediterranean striped dolphins (Stenella coeruleoalba). Systemic infection caused by DMV in the Mediterranean has been reported only during these outbreaks. RESULTS: We report the infection of five striped dolphins (Stenella coeruleoalba) stranded on the Spanish Mediterranean coast of Valencia after the last DMV outbreak that ended in 2011. Animal 1 stranded in late 2011 and Animal 2 in 2012. Systemic infection affecting all tissues was found based on histopathology and positive immunohistochemical and polymerase chain reaction positive results. Animal 3 stranded in 2014; molecular and immunohistochemical detection was positive only in the central nervous system. Animals 4 and 5 stranded in 2015, and DMV antigen was found in several tissues. Partial sequences of the DMV phosphoprotein (P), nucleoprotein (N), and hemagglutinin (H) genes were identical for Animals 2, 3, 4, and 5, and were remarkably different from those in Animal 1. The P sequence from Animal 1 was identical to that of the DMV strain that caused the epizootic of 2011 in the Spanish Mediterranean. The corresponding sequence from Animals 2-5 was identical to that from a striped dolphin stranded in 2011 on the Canary Islands and to six dolphins stranded in northeastern Atlantic of the Iberian Peninsula. CONCLUSIONS: These results suggest the existence of an endemic infection cycle among striped dolphins in the Mediterranean that may lead to occasional systemic disease presentations outside epizootic periods. This cycle involves multiple pathogenic viral strains, one of which may have originated in the Atlantic Ocean.

Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed.

First molecular determination of herpesvirus from two mysticete species stranded in the Mediterranean Sea.

BACKGROUND: Herpesvirus can infect a wide range of animal species: mammals, birds, reptiles, fish, amphibians and bivalves. In marine mammals, several alpha- and gammaherpesvirus have been identified in some cetaceans and pinnipeds species. To date, however, this virus has not been detected in any member of the Balaenoptera genus. CASE PRESENTATION: Herpesvirus was determined by molecular methods in tissue samples from a male fin whale juvenile (Balaenoptera physalus) and a female common minke whale calf (Balaenoptera acutorostrata) stranded on the Mediterranean coast of the Region of Valencia (Spain). Samples of skin and penile mucosa from the fin whale and samples of skin, muscle and central nervous system tissue from the common minke whale tested positive for herpesvirus based on sequences of the DNA polymerase gene. Sequences from fin whale were identical and belonged to the Alphaherpesvirinae subfamily. Only members of the Gammaherpesvirinae subfamily were amplified from the common minke whale, and sequences from the muscle and central nervous system were identical. Sequences in GenBank most closely related to these novel sequences were viruses isolated from other cetacean species, consistent with previous observations that herpesviruses show similar phylogenetic branching as their hosts. CONCLUSIONS: To our knowledge, this is the first molecular determination of herpesvirus in the Balaenoptera genus. It shows that herpesvirus should be included in virological evaluation of these animals.

Novel adenovirus detected in captive bottlenose dolphins (Tursiops truncatus) suffering from self-limiting gastroenteritis.

BACKGROUND: Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION: A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION: These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations.

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens).

BACKGROUND: Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION: In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION: To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.

Platelet phosphatidylserine exposure and microparticle production as health bioindicators in marine mammals.

In human medicine, various pathologies, including decompression sickness, thrombocytopenia, and rheumatoid arthritis, have been linked to changes in cellular microparticles (MP) formation, particularly platelet microparticles (PMP). Similar disorders in marine mammals might be attributed to anthropogenic threats or illnesses, potentially impacting blood PMP levels. Thus, detecting platelet phosphatidylserine (PS) exposure and PMP formation could serve as a crucial diagnostic and monitoring approach for these conditions in marine mammals. Our group has developed a methodology to assess real-time PS exposure and PMP formation specifically tailored for marine mammals. This method, pioneered in species such as bottlenose dolphins, beluga whales, walruses, and California sea lions, represents a novel approach with significant implications for both clinical assessment and further research into platelet function in these animals. The adapted methodology for evaluating PS exposure and PMP formation in marine mammals has yielded promising results. By applying this approach, we have observed significant correlations between alterations in PMP levels and specific pathologies or environmental factors. These findings underscore the potential of platelet function assessment as a diagnostic and monitoring tool in marine mammal health. The successful adaptation and application of this methodology in marine mammals highlight its utility for understanding and managing health concerns in these animals.

Physiological values of phagocytic capacity in marine mammals and alterations during pathological situations.

The study of the immune function in marine mammals is essential to understand their physiology and can help to improve their welfare in the aquariums. Dedicating efforts to studying marine mammal physiology, pathophysiology, and implementing new diagnostic and therapeutic tools promote progress towards preventive medicine in aquariums by facilitating early detection and treatment of diseases. However, biological and clinical research on marine mammals is currently very limited due to difficult access to these species and their biological samples. With this objective, our group has adapted to marine mammals a commercially available assay routinely used to evaluate the phagocytic capacity of monocytes and granulocytes in human whole blood samples. We adapted IngoflowEx kit to bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), walruses (Odobenus rosmarus), Patagonian sea lions (Otaria flavescens), and harbor (Phoca vitulina). In this paper, we report the modifications carried out on the original protocol for their correct functioning in marine mammals. We obtained physiological values of phagocytic capacity in each species after repeated sampling for 4 years in various individuals of each species. Specific results revealed that the % phagocytic cells that ingested E.coli in bottlenose dolphins were 59.6 ± 1.27, in walruses 62.6 ± 2.17, in sea lions 57.5 ± 4.3, and in beluga whales 61.7 ± 1.4. In the case of the % phagocytic cells producing respiratory burst in bottlenose dolphins were 34.2 ± 3.6, in walruses 36.3 ± 4.3, in sea lions 40.8 ± 10.2, and in beluga whales 26.3 ± 3.7. These preliminary results can be used as a reference to detect alterations in phagocytic capacity either by immunosuppression or by exacerbation of the response in infectious inflammatory processes. Clinical applicability of the assay was verified in two clinical cases in which Ingoflow was useful to detect immune alterations in two diseased individuals, before and after the onset of clinical signs.

Unusual striped dolphin mass mortality episode related to cetacean morbillivirus in the Spanish Mediterranean sea.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the two dolphin morbillivirus (DMV) epizootics of 1990-1992 and 2006-2008, which affected Mediterranean striped dolphins (Stenella coeruleoalba). Between March and April 2011, the number of strandings on the Valencian Community coast (E Spain) increased. CASE PRESENTATION: Necropsy and sample collection were performed in all stranded animals, with good state of conservation. Subsequently, histopathology, immunohistochemistry, conventional reverse transcription polymerase chain reaction (RT-PCR) and Universal Probe Library (UPL) RT-PCR assays were performed to identify Morbillivirus. Gross and microscopic findings compatible with CeMV were found in the majority of analyzed animals. Immunopositivity in the brain and UPL RT-PCR positivity in seven of the nine analyzed animals in at least two tissues confirmed CeMV systemic infection. Phylogenetic analysis, based on sequencing part of the phosphoprotein gene, showed that this isolate is a closely related dolphin morbillivirus (DMV) to that responsible for the 2006-2008 epizootics. CONCLUSION: The combination of gross and histopathologic findings compatible with DMV with immunopositivity and molecular detection of DMV suggests that this DMV strain could cause this die-off event.

Two cases of pseudohermaphroditism in loggerhead sea turtles Caretta caretta.

Two juvenile (curved carapace lengths: 28 and 30 cm) loggerhead sea turtles Caretta caretta with precocious male external characteristics were admitted to the ARCA del Mar rescue area at the Oceanogràfic Aquarium in Valencia, Spain, in 2009 and 2010. Routine internal laparoscopic examination and subsequent histopathology confirmed the presence of apparently healthy internal female gonads in both animals. Extensive tissue biopsy and hormone induction assays were consistent with female sex. To the best of our knowledge, this is the first report of pseudohermaphroditism in loggerhead sea turtles based on sexual external characteristics and internal laparoscopic examination. Our findings suggest that the practice of using external phenotypical characteristics as the basis for gender identification in sea turtles should be reevaluated. Future research should focus on detecting more animals with sexual defects and their possible effects on the sea turtle population.

A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020.

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Molecular and Serological Studies on Potential SARS-CoV-2 Infection among 43 Lemurs under Human Care-Evidence for Past Infection in at Least One Individual.

In the setting of the recent COVID-19 pandemic, transmission of SARS-CoV-2 to animals has been reported in both domestic and wild animals and is a matter of concern. Given the genetic and functional similarities to humans, non-human primates merit particular attention. In the case of lemurs, generally considered endangered, they are believed to be susceptible to SARS-CoV-2 infection. We have conducted a study for evidence of SARS-CoV-2 infection among the 43 lemurs of Mundomar, a zoological park in Benidorm, Spain. They belong to two endangered lemur species, 23 black-and-white ruffed lemurs (Varecia variegata) and 20 ring-tailed lemurs (Lemur catta). Health assessments conducted in 2022 and 2023 included molecular analyses for SARS-CoV-2 RNA of oral and rectal swabs using two different RT-qPCR assays, always with negative results for SARS-CoV-2 in all animals. The assessment also included serological testing for antibodies against the receptor-binding domain (RBD) of the spike protein (S) of SARS-CoV-2, which again yielded negative results in all animals except one black-and-white ruffed lemur, supporting prior infection of that animal with SARS-CoV-2. Our data, while not indicating a high susceptibility of lemurs to SARS-CoV-2 infection, show that they can be infected, adding to the existing information body on potential ways for SARS-CoV-2 virus spreading in zoos, highlighting the need for animal surveillance for the virus.

The Finding of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) in a Wild Eurasian River Otter (Lutra lutra) Highlights the Need for Viral Surveillance in Wild Mustelids.

Animals have been involved in the three known outbreaks of severe respiratory syndromes due to coronaviruses (years 2005, 2012, and 2019). The pandemic nature of the SARS-CoV-2 outbreak increases the likelihood of infection from humans of susceptible animal species that, thus, could become secondary viral hosts and even disease reservoirs. We present evidence of spillover infection of wild mustelids by reporting the presence of SARS-CoV-2 in a Eurasian river otter found near a water reservoir in the Valencian Community (Spain). We detected the virus using two different commercial RTqPCR assays on RNA extracted from the nasopharynx (swabbing) and from lung tissue and mediastinal lymph node homogenates. The corresponding samples from two additional otters from distant sites tested negative in identical assays. The diagnosis in the positive otter was confirmed by two-tube RT-PCR assay in which RNA was first retrotranscribed, and then specific regions of the spike (S), nucleocapsid (N), and ORF10 genes were separately amplified from the produced cDNA, followed by electrophoretic visualization and Sanger sequencing. The sequences of the amplified products revealed some non-synonymous changes in the N and ORF10 partial sequences, relative to the consensus sequence. These changes, identified already in human patient samples, point to human origin of the virus, although their specific combination was unique. These findings, together with our previous report of SARS-CoV-2 infection of feral American mink, highlight the need for SARS-CoV-2 surveillance of wild or feral mustelids to evaluate the risk that these animals could become SARS-CoV-2 reservoirs.

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic.

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

First case of erysipelas in a free-ranging bottlenose dolphin (Tursiops truncatus) stranded in the Mediterranean Sea.

An adult female bottlenose dolphin (Tursiops truncatus) stranded alive and subsequently died several minutes later on the Mediterranean coast of Spain on 14 July 2010. Clinical examination revealed foam through the blowhole and rales upon lung auscultation. On venipuncture, the blood was abnormally dense and dark. Hematological and biochemical abnormalities included dehydration, leukocytosis (48 600 leukocytes microl(-1)) characterized by neutrophilia (48 200 neutrophils microl(-1)), and elevated bilirubin (4.38 mg dl(-1)), alanine aminotransferase (382.3 U l(-1)), aspartate aminotransferase (1449.3 U l(-1)), lactate dehydrogenase (1631.3 U l(-1)), and creatine kinase (404.7 U l(-1)). The most relevant findings of the gross examination were rhomboid-shaped skin lesions, stable froth in the trachea, pulmonary congestion, abnormally thick and rough pleura with adhesions, edematous and congestive superficial cervical and tracheobronchial lymph nodes, red-tinged urine, and severe brain congestion. Histopathology of the kidney, lung, skin, and brain revealed multisystemic intravascular bacterial emboli. Samples of skin, brain, and lung were cultured on Columbia blood agar under both aerobic and anaerobic conditions, and pure and heavy bacterial cultures were obtained from skin and brain samples. The microorganism isolated was Gram-positive, catalase-negative, facultatively anaerobic, and rod-shaped. The isolates were identified as Erysipelothrix rhusiopathiae by the API Coryne biochemical system. Based on the gross and microscopic findings, a diagnosis of acute E. rhusiopathiae septicemia was made. To the best of our knowledge, this is the first report of E. rhusiopathiae septicemia in a free-ranging bottlenose dolphin.

First Description of SARS-CoV-2 Infection in Two Feral American Mink (Neovison vison) Caught in the Wild.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, is considered a pathogen of animal origin that is mainly transmitted from human to human. Several animal species can be naturally or experimentally infected by SARS-CoV-2, with compelling evidence that mink is highly susceptible to SARS-CoV-2 infection. Human-to-mink infection cases have been reported and there are also suggestions that mink-to-human infection occurs. Mink infections have been reported to date only on fur farms, except for one infected free- ranging wild mink near a Utah (USA) fur farm, which suggests a transmission pathway from farms to wild mink. We now report the detection of SARS-CoV-2 in 2 of 13 feral dark brown American mink (Neovison vison) trapped in the Valencian Community (Eastern Spain), during an invasive species trapping campaign. They were trapped in riverbeds in sparsely inhabited rural areas known to harbor self-sustained feral mink populations. The closest fur farm is about 20 km away. SARS-CoV-2 RNA was detected by two-step RT-PCR in these animals' mesenteric lymph nodes and was confirmed by sequencing a 397-nucleotide amplified region of the S gene, yielding identical sequences in both animals. A molecular phylogenetic analysis was run on this sequence, which was found to correspond to the consensus SARS-CoV-2 sequence from Wuhan. Our findings appear to represent the first example of SARS-CoV-2 acquired in the wild by feral mink in self-sustained populations.

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

Circulation of a novel strain of dolphin morbillivirus (DMV) in stranded cetaceans in the Mediterranean Sea.

Dolphin morbillivirus (DMV) has been responsible for several outbreaks of systemic infection and has resulted in cetacean strandings in the Mediterranean. In August-October 2016, seven striped dolphins (Stenella coeruleoalba) stranded on the Sicilian coastline (Italy) tested positive for DMV. Tissue samples from brain, lung, pulmonary lymph nodes, heart, spleen, liver, stomach, intestine, kidneys and urinary bladder, as well as blowhole swabs, were collected during necropsy for molecular diagnostics and pathology studies. Extracted tissue RNA was screened for DMV by real-time reverse transcription polymerase chain reaction (PCR). Some tissues exhibited microscopic lesions that were consistent with DMV infection on histopathological and immunohistochemical grounds. Conventional reverse transcription PCR to target partial nucleoprotein and phosphoprotein genes yielded sequences used to genetically characterize the associated DMV strain. DMV RNA was detected by both PCR assays in all tested tissues of the seven dolphins, which suggests systemic infections, but was absent from another dolphin stranded on the Sicilian coastline during the same period. The partial phosphoprotein and nucleoprotein gene sequences from the positive dolphins were 99.7% and 99.5% identical, respectively, to the DMV sequences recently observed in cetaceans stranded on the Spanish Mediterranean. Our study suggests that this DMV strain is circulating in the Mediterranean.

Phylogenomic analysis of the complete sequence of a gastroenteritis-associated cetacean adenovirus (bottlenose dolphin adenovirus 1) reveals a high degree of genetic divergence.

Adenoviruses are common pathogens in vertebrates, infecting a wide range of hosts, but only having rarely been detected and correlated with disease in cetaceans. This article describes the first complete genomic sequence of a cetacean adenovirus, bottlenose dolphin adenovirus 1 (BdAdV-1), detected in captive bottlenose dolphin population (Tursiops truncatus) suffering from self-limiting gastroenteritis. The complete genome sequence of BdAdV-1 was recovered from data generated by high-throughput sequencing and validated by Sanger sequencing. The genome is 34,080bp long and has 220 nucleotides long inverted terminal repeats. A total of 29 coding sequences were identified, 26 of which were functionally annotated. Among the unusual features of this genome is a remarkably long 4380bp E3 ORF1, that displays no sequence homology with the corresponding E3 regions of other adenoviruses. In addition, the fiber protein only has 26% identity with fiber proteins described in other adenoviruses. Three hypothetical proteins were predicted. The phylogenetic analysis indicates that the closest known relative to BdAdV-1 is an adenovirus detected in bottlenose dolphin (KR024710), with an amino acid sequence identity between 36 and 79% depending on the protein. Based on the phylogenic analysis, the BdAdV-1 appears to have co-evolved with its host. The results indicate that BdAdV-1 belongs to the Mastadenovirus genus of the Adenoviridae family, however, it is clearly different from other adenoviruses, especially in the 3'-end of the viral genome. The high degree of sequence divergence suggests that BdAdV-1 should be considered as a novel species in the Mastadenovirus genus. The study also demonstrates the usefulness of high-throughput sequencing to obtain full-length genomes of genetically divergent viruses.