Novel herpesvirus in the critically endangered Galapagos pink land iguana.

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

Molecular survey of selected viruses in Pudus (Pudu puda) in Chile revealing first identification of caprine herpesvirus-2 (CpHV-2) in South American ungulates.

Viral diseases jeopardize the health of wildlife in Chile. However, this country lacks health surveillance programs that allow for defining preventive measures to tackle such diseases. The objective of this study was to determine the occurrence and the genetic diversity of pestivirus, herpesvirus and adenovirus in pudus from Chile. Blood samples from wild (n=34) and captive (n=32) pudus were collected between 2011 and 2019 and analyzed through consensus PCR. All the samples were negative to pestivirus and adenovirus. Herpesvirus was confirmed in four captive, and one wild pudu. All four zoo animals share the same sequence for both polymerase and glycoprotein genes. Both sequences share a 100% identity with caprine herpesvirus-2, classifying them in the same cluster as the Macavirus group. In turn, novel sequences of the polymerase and glycoprotein B genes were obtained from the wild pudu. Our study reports the first evidence of CpHV-2 infection in Chile and South American ungulate populations. Further research will be necessary to assess the pathogenicity of CpHV-2 in this species. It is also urgently recommended that molecular, serological and pathological screening should be conducted in Chilean wild and captive pudus to understand the impact of the herpesvirus on their populations.

Adenovirus surveillance in wild carnivores from Brazil.

Landscape transformation favors the spread of new pathogens that can be shared between domestic and wild animals. Certain adenoviruses (e.g., canine adenovirus 1 and 2, family Adenoviridae) can infect domestic and wild carnivores. In domestic canids, these viruses are associated with hepatic and respiratory diseases (among others). Nevertheless, information regarding adenovirus pathogenicity and molecular features in wild carnivores is still limited. Herein we surveyed adenovirus in free-ranging carnivores from Brazil. Total DNA was extracted from and subsequently tested by a nested panPCR in spleen and/or lung of 52 carnivores, representing species of the following families: Canidae (n = 4), Felidae (n = 3), Mustelidae (n = 2) and Procyonidae (n = 2). The obtained sequences were compared to others available at GenBank. Available tissue samples from the positive cases were evaluated histopathologically. One out of 52 (1.9%, CI 95%, 0.0-5.7%) carnivores was positive; a roadkilled ocelot (Leopardus pardalis). The obtained sequence presented a low deduced amino acid (78.1%) similarity with the closest adenovirus, identified in a pinniped from the United States of America. This fact and its detection in a novel host suggest it may be representative of a novel species and denominated ocelot adenovirus 1. None of the gross and microscopic findings of the positive case were associated with adenovirus. To the authors' knowledge, this is the first report of adenovirus in wild felids of South America and the second worldwide. Further studies are necessary to assess the epidemiology and potential pathogenicity of this agent in wild carnivores.

Health assessments uncover novel viral sequences in five species of Galapagos tortoises.

Emerging infectious diseases (EIDs) have been reported as causes of morbidity and mortality in free-living animal populations, including turtles and tortoises, and they have even resulted in species extinctions, with human activities contributing to the spread of many of these diseases. In the Galapagos, giant tortoises are endangered due to habitat change, invasive species, and other human impacts; however, the impact of EIDs on Galapagos tortoise conservation remains understudied. To fill this gap, we conducted health assessments of five tortoise species from the islands of Santa Cruz, Isabela and Española. We performed health evaluations of 454 animals and PCR testing for pathogens known to be relevant in other tortoise species. We identified two novel sequences of adenoviruses and four of herpesviruses. Based on alignments of the DNA polymerase gene and maximum likelihood phylogenetic analyses, we found both novel adenoviruses to be most closely related to red footed tortoise adenovirus 2, by nucleotide sequence and red footed tortoise adenovirus 1, based on amino acid sequence. Three of the herpesvirus sequences translated into the same deduced amino acid sequence; therefore, they may be considered the same viral species, closely related to terrapene herpesvirus 2. The fourth herpesvirus sequence was highly divergent from any sequence previously detected and is related to an eagle owl herpesvirus based on nucleotide sequence and to loggerhead oro-cutaneous herpesvirus based on amino acids. These novel viruses may be pathogenic for giant tortoises under specific conditions (e.g., stress). Continued screening is crucial to determine if these viruses play a role in tortoise fitness, morbidity and survival. This information allows us to provide recommendations to the Galapagos National Park Directorate and other institutions to improve the management of these unique species both in Galapagos and globally, and for tortoise reintroduction plans throughout the archipelago.

Highly divergent herpesviruses in threatened river dolphins from Brazil.

River dolphins are a highly threatened polyphyletic group comprised of four odontocete families: Iniidae, Pontoporiidae, Lipotidae, and Platanistidae, the first two endemic to South America. To address the knowledge gap regarding infectious agents in this cetacean group, we surveyed the presence of herpesviruses by PCR in skin and/or blood samples of live-captured Amazon (Inia geoffrensis, n = 25) and Bolivian (Inia boliviensis, n = 22) river dolphins of the Amazon basin and in selected tissue samples of franciscanas (Pontoporia blainvillei, n = 27) stranded or bycaught in southeastern Brazil. Additionally, available franciscana tissue samples were examined by histopathology. Herpesvirus DNA was amplified in 13 Bolivian river dolphins (59.1%, 95% CI 38.5-79.6%) and 14 franciscanas (51.9%, 95% CI 33.0-70.7%). All Amazon river dolphins were herpesvirus-negative. Two different herpesviruses were found in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and/or skin samples of all positive individuals and a novel alphaherpesvirus in the skin of one animal. A new gammaherpesvirus was found in several franciscana samples-the first herpesvirus recorded in Pontoporiidae. Intranuclear inclusion bodies consistent with herpesvirus were observed in the lymph node of one franciscana. The high divergence among the obtained herpesviruses and those previously described can be explained by viral-host coevolution, and by the fact that these populations are fairly isolated.

Neoplasms and novel gammaherpesviruses in critically endangered captive European minks (Mustela lutreola).

The European mink (Mustela lutreola) is a riparian mustelid, considered one of the most endangered carnivores in the world. Alpha, beta and gammaherpesviruses described in mustelids have been occasionally associated with different pathological processes. However, there is no information about the herpesviruses species infecting European minks. In this study, 141 samples of swabs (oral, conjunctival, anal), faeces and tissues from 23 animals were analysed for herpesvirus (HV) using a pan-HV-PCR assay. Two different, potentially novel, gammaherpesvirus species were identified in 12 samples from four animals (17.3%), and tentatively named Mustelid gammaherpesvirus-2 (MUGHV-2) and MuGHV-3. Gross examination was performed on dead minks (n = 11), while histopathology was performed using available samples from HV-positive individuals (n = 2), identifying several neoplasms, including B-cell lymphoma (identified by immunohistochemistry) with intralesional syncytia and intranuclear inclusion bodies characteristic of HV (n = 1), pulmonary adenocarcinoma (n = 1), and biliary (n = 1) and preputial (n = 1) cystadenomas, as well as other lesions (e.g., axonal vacuolar degeneration [n = 2] and neuritis [n = 1]). Viral particles, consistent with HVs, were observed by electron microscopy in the mink with neural lymphoma and inclusion bodies. This is the first description of neoplasms and concurrent gammaherpesvirus infection in European minks. The pathological, ultrastructural and PCR findings (MuGHV-2) in the European mink with lymphoma strongly suggest a potential role for this novel gammaherpesvirus in its pathogenesis, as it has been reported in other HV-infected species with lymphoma. The occurrence of neural lymphoma with intralesional syncytia and herpesviral inclusions is, however, unique among mammals. Further research is warranted to elucidate the potential oncogenic properties of gammaherpesviruses in European mink and their epidemiology in the wild population.

Fatal pulmonary parafilaroidiasis in a free-ranging subantarctic fur seal (Arctocephalus tropicalis) coinfected with two gammaherpesviruses and Sarcocystis sp.

A juvenile subantarctic fur seal (Arctocephalus tropicalis) found dead in Santa Catarina state, southern Brazil, presented with disseminated verminous pneumonia due to Parafilaroides sp. A concomitant infection with two different gammaherpesviruses was identified by PCR in different tissues; one of them possibly a novel species (tentatively named Otariid herpesvirus 7). Sarcocystis sp. DNA was identified molecularly in skeletal muscle samples with intrasarcoplasmic bradyzoites and no apparent tissue response. All analyzed samples (mandibular, laryngeal, tracheal, and mesenteric lymph nodes, and lung) were PCR-negative for Brucella spp. The most likely cause of death was severe pulmonary parafilaroidiasis. The pathogenic role of the gammaherpesviruses in several of the tissues was not evident. This study describes the pathogenicity of Parafilaroides sp. in a subantarctic fur seal, widens the host range of herpesvirus in pinnipeds, and reports the first molecular identification of Sarcocystis sp. in this species.

Fatal pulmonary parafilaroidiasis in a free-ranging subantarctic fur seal (Arctocephalus tropicalis) coinfected with two gammaherpesviruses and Sarcocystis sp / Parafilaroidose pulmonar fatal em um lobo-marinho-subantártico (Arctocephalus tropicalis) de vida livre coinfectado com dois gammaherpesvírus e Sarcocystis sp

Abstract A juvenile subantarctic fur seal (Arctocephalus tropicalis) found dead in Santa Catarina state, southern Brazil, presented with disseminated verminous pneumonia due to Parafilaroides sp. A concomitant infection with two different gammaherpesviruses was identified by PCR in different tissues; one of them possibly a novel species (tentatively named Otariid herpesvirus 7). Sarcocystis sp. DNA was identified molecularly in skeletal muscle samples with intrasarcoplasmic bradyzoites and no apparent tissue response. All analyzed samples (mandibular, laryngeal, tracheal, and mesenteric lymph nodes, and lung) were PCR-negative for Brucella spp. The most likely cause of death was severe pulmonary parafilaroidiasis. The pathogenic role of the gammaherpesviruses in several of the tissues was not evident. This study describes the pathogenicity of Parafilaroides sp. in a subantarctic fur seal, widens the host range of herpesvirus in pinnipeds, and reports the first molecular identification of Sarcocystis sp. in this species.

Resumo Um lobo-marinho-subantártico (Arctocephalus tropicalis) juvenil foi achado morto no Estado de Santa Catarina, sul do Brasil, apresentando pneumonia parasitária disseminada por Parafilaroides sp. Infecção concomitante por dois gammaherpesvírus diferentes foi identificada pela PCR em diversos tecidos, um desses herpesvírus possivelmente uma nova espécie (denominada provisoriamente Otariid herpesvirus 7). DNA de Sarcocystis sp. foi identificado molecularmente em amostras de músculo esquelético que apresentavam bradizoítos intra-sarcoplasmáticos sem aparente resposta tecidual. Todas as amostras analisadas (linfonodo mandibular, laríngeo, traqueal e mesentérico, e pulmão) pela PCR para Brucella spp. foram negativas. A causa mais provável da morte do animal foi parafilaroidose pulmonar severa. O papel patogénico dos gammaherpesvírus em vários tecidos não foi evidente. Este estudo descreve a patogenicidade de Parafilaroides sp. em um lobo-marinho-subantártico, amplia a variedade de hospedeiros de herpesvírus em pinípedes e reporta a primeira identificação molecular de Sarcocystis sp. para essa espécie.

Identification of Novel Gammaherpesviruses in a South American Fur Seal ( Arctocephalus australis) with Ulcerative Skin Lesions.

There are few studies on pathogens affecting free-ranging pinnipeds from South America. We employed molecular techniques to identify a gammaherpesvirus infection by two putative novel herpesvirus species: Otariid herpesvirus 5 (OtHV-5), possibly associated with ulcerative cutaneous lesions, and Otariid herpesvirus 6 (OtHV-6) in a wild South American fur seal ( Arctocephalus australis) that stranded alive in Santa Catarina state, southern Brazil. Here we provide new information regarding pinniped herpesviruses, important for the design of future disease surveillance studies.

Identification of a gammaherpesvirus belonging to the malignant catarrhal fever group of viruses in Pyrenean chamois (Rupicapra p. pyrenaica).

High prevalence (46 %) of a gammaherpesvirus was confirmed by molecular detection in the lungs of hunted Pyrenean chamois. The partial glycoprotein B sequence up to the DNA polymerase gene showed 96.6 % nucleotide sequence identity to the Rupicapra rupicapra gammaherpesvirus 1 and 81.5 % to ovine herpesvirus 2. This novel sequence clusters within sequences derived from the malignant catarrhal fever group of viruses, and the corresponding virus is tentatively named Rupicapra pyrenaica gammaherpesvirus 1 (RpHV-1). No specific histological lesions were associated with RpHV-1, nor were any detrimental effects on host health. The epidemiological, phylogenetic and histopathological results suggest that Pyrenean chamois is the natural host of RpHV-1.

Patterns of Exposure of Iberian Wolves (Canis lupus) to Canine Viruses in Human-Dominated Landscapes.

Wildlife inhabiting human-dominated landscapes is at risk of pathogen spill-over from domestic species. With the aim of gaining knowledge in the dynamics of viral infections in Iberian wolves (Canis lupus) living in anthropized landscapes of northern Spain, we analysed between 2010 and 2013 the samples of 54 wolves by serology and polymerase chain reaction (PCR) for exposure to four pathogenic canine viruses: canine distemper virus (CDV), canine parvovirus-2 (CPV), canine adenovirus 1 and 2 (CAV-1 and CAV-2) and canine herpesvirus. Overall, 76% of the studied wolves presented evidence of exposure to CPV (96% by HI, 66% by PCR) and 75% to CAV (75% by virus neutralization (VN), 76% by PCR, of which 70% CAV-1 and 6% CAV-2). This represents the first detection of CAV-2 infection in a wild carnivore. CPV/CAV-1 co-infection occurred in 51% of the wolves. The probability of wolf exposure to CPV was positively and significantly correlated with farm density in a buffer zone around the place where the wolf was found, indicating that rural dogs might be the origin of CPV infecting wolves. CPV and CAV-1 appear to be enzootic in the Iberian wolf population, which is supported by the absence of seasonal and inter-annual variations in the proportion of positive samples detected. However, while CPV may depend on periodical introductions by dogs, CAV-1 may be maintained within the wolf population. All wolves were negative for exposure to CDV (by VN and PCR) and CHV (by PCR). The absence of acquired immunity against CDV in this population may predispose it to an elevated rate of mortality in the event of a distemper spill-over via dogs.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

Molecular identification of a novel gammaherpesvirus in the endangered Darwin's fox (Lycalopex fulvipes).

We report the detection and characterization of a novel gammaherpesvirus in the critically endangered Darwin's fox (Lycalopex fulvipes; syn. Pseudalopex fulvipes) on Chiloé Island, Chile. Out of 28 analysed blood samples stored in alcohol, four were positive for this herpesvirus using a previously described pan-herpesvirus PCR assay targeting the herpesvirus DNA polymerase. Positive samples were subsequently characterized by means of a PCR targeting a 500 bp fragment of the glycoprotein B of the gammaherpesviruses. This novel herpesvirus was most closely related to other gammaherpesviruses from terrestrial carnivores, and is tentatively named Darwin's fox gammaherpesvirus. No apparent lesions were observed in the surveyed foxes. This is the first report of a gammaherpesvirus infecting a canid worldwide, and also of one infecting a carnivore from South America.

Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity.

We report the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosis-like lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed.