Spleen and lung virome analysis of South American fur seals (Arctocephalus australis) collected on the southern Brazilian coast.

South American fur seals (Arctocephalus australis) are believed to reach the coast of Rio Grande do Sul (RS) through sea currents. They live in colonies and are frequently found resting on the beach. However, it is also common to find dead pinnipeds on beaches, sharing the environment with humans, domestic animals and other wild species on the coast and facilitating the transmission of pathogens. In the present study, a metagenomic approach was applied to evaluate the viral diversity in organs of fur seals found deceased along the coast of the state of RS, southern Brazil. The lungs and spleens of 29 animals were collected, macerated individually, pooled separately (one pool for lungs and another for spleens) and sequenced using the Illumina MiSeq platform. Sequences more closely related to members of the Anelloviridae and Circoviridae families were detected. Nine putative new species of anellovirus and one putative new genus, named Nitorquevirus, were described. Additionally, the circovirus sequences found in the lungs of A. australis have a common ancestor with PCV3, a proposed swine pathogen. Our study expanded the knowledge about viral communities in pinnipeds and could be useful for monitoring new viruses and potential viral sharing among wildlife, domestic animals, and humans.

Hemagglutinin Stability and Its Impact on Influenza A Virus Infectivity, Pathogenicity, and Transmissibility in Avians, Mice, Swine, Seals, Ferrets, and Humans.

Genetically diverse influenza A viruses (IAVs) circulate in wild aquatic birds. From this reservoir, IAVs sporadically cause outbreaks, epidemics, and pandemics in wild and domestic avians, wild land and sea mammals, horses, canines, felines, swine, humans, and other species. One molecular trait shown to modulate IAV host range is the stability of the hemagglutinin (HA) surface glycoprotein. The HA protein is the major antigen and during virus entry, this trimeric envelope glycoprotein binds sialic acid-containing receptors before being triggered by endosomal low pH to undergo irreversible structural changes that cause membrane fusion. The HA proteins from different IAV isolates can vary in the pH at which HA protein structural changes are triggered, the protein causes membrane fusion, or outside the cell the virion becomes inactivated. HA activation pH values generally range from pH 4.8 to 6.2. Human-adapted HA proteins tend to have relatively stable HA proteins activated at pH 5.5 or below. Here, studies are reviewed that report HA stability values and investigate the biological impact of variations in HA stability on replication, pathogenicity, and transmissibility in experimental animal models. Overall, a stabilized HA protein appears to be necessary for human pandemic potential and should be considered when assessing human pandemic risk.

Updating the influenza virus library at Hokkaido University -It's potential for the use of pandemic vaccine strain candidates and diagnosis.

Genetic reassortment of influenza A viruses through cross-species transmission contributes to the generation of pandemic influenza viruses. To provide information on the ecology of influenza viruses, we have been conducting a global surveillance of zoonotic influenza and establishing an influenza virus library. Of 4580 influenza virus strains in the library, 3891 have been isolated from over 70 different bird species. The remaining 689 strains were isolated from humans, pigs, horses, seal, whale, and the environment. Phylogenetic analyses of the HA genes of the library isolates demonstrate that the library strains are distributed to all major known clusters of the H1, H2 and H3 subtypes of HA genes that are prevalent in humans. Since past pandemic influenza viruses are most likely genetic reassortants of zoonotic and seasonal influenza viruses, a vast collection of influenza A virus strains from various hosts should be useful for vaccine preparation and diagnosis for future pandemics.

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.

Novel anelloviruses identified in buccal swabs of Antarctic fur seals.

Viral diversity associated with Antarctic wildlife remains poorly studied. Nonetheless, over the past 5 years, there has been a concerted effort using viral metagenomics approaches to identify and characterize viruses associated with Antarctic pinniped and avian species. Here we used a viral metagenomics approach to identify circular DNA viruses in buccal swab samples from Antarctic fur seals (Arctocephalus gazella) breeding on Livingston Island, Antarctica during the 2016/2017 field season. We identified two new lineages of anelloviruses, torque teno Arctocephalus gazella virus (TTAgV)-1 (2083 nts) and -2 (2127-2143 nts), which are most closely related to and cluster with a previously identified anellovirus associated with California sea lions (Zalophus californianus) sharing ~ 60% genome-wide pairwise identity. The ORF1 of TTAgVs share 26-41% amino acid similarity to the ORF1 of other previously identified pinniped-associated anelloviruses.

Discovery of fur seal feces-associated circular DNA virus in swine feces in Japan.

Fur seal feces-associated circular ssDNA virus (FSfaCV) was discovered in a pig for the first time in Japan using a next-generation sequencer with duplex-specific nuclease. Full genome of the virus showed approximately 92% similarity to FSfaCVs from New Zealand fur seals. Furthermore, we investigated the prevalence of the ssDNA virus in 85 piglets in Japan, and 65 piglets were positive (76%) for the virus.

Molecular Detection of Circovirus and Adenovirus in Feces of Fur Seals (Arctocephalus spp.).

In some regions, little is known about exposure to viruses in coastal marine mammals. The present study aimed to detect viral RNA or DNA in 23 free-ranging fur seals on the northern coastline of Rio Grande do Sul State, Brazil. Polymerase chain reaction was used to detect nucleic acids of circoviruses, adenoviruses, morbilliviruses, vesiviruses, and coronaviruses in the feces from twenty-one South American fur seals (Arctocephalus australis) and two Subantarctic fur seals (A. tropicalis). Adenovirus DNA fragments were detected in two South American fur seals; nucleotide sequences of these fragments revealed a high degree of similarity to human adenovirus type C. Circovirus DNA fragments were detected in six animals of the same species. Two were phylogenetically similar to the Circovirus genus, whereas the other four nucleotide fragments showed no similarity to any of the known genera within the family Circoviridae. RNA fragments indicating the presence of coronavirus, vesivirus, and morbillivirus were not detected. These findings suggest that adenoviruses and circoviruses are circulating in fur seal populations found along the coast of Rio Grande do Sul State, Brazil.

Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals.

The Brazilian South coast seasonally hosts numerous marine species, observed particularly during winter months. Some animals, including fur seals, are found dead or debilitated along the shore and may harbor potential pathogens within their microbiota. In the present study, a metagenomic approach was performed to evaluate the viral diversity in feces of fur seals found deceased along the coast of the state of Rio Grande do Sul. The fecal virome of two fur seal species was characterized: the South American fur seal (Arctocephalus australis) and the Subantarctic fur seal (Arctocephalus tropicalis). Fecal samples from 10 specimens (A. australis, n = 5; A. tropicalis, n = 5) were collected and viral particles were purified, extracted and amplified with a random PCR. The products were sequenced through Ion Torrent and Illumina platforms and assembled reads were submitted to BLASTx searches. Both viromes were dominated by bacteriophages and included a number of potentially novel virus genomes. Sequences of picobirnaviruses, picornaviruses and a hepevirus-like were identified in A. australis. A rotavirus related to group C, a novel member of the Sakobuvirus and a sapovirus very similar to California sea lion sapovirus 1 were found in A. tropicalis. Additionally, sequences of members of the Anelloviridae and Parvoviridae families were detected in both fur seal species. This is the first metagenomic study to screen the fecal virome of fur seals, contributing to a better understanding of the complexity of the viral community present in the intestinal microbiota of these animals.

A NOVEL GAMMAHERPESVIRUS IN NORTHERN FUR SEALS (CALLORHINUS URSINUS) IS CLOSELY RELATED TO THE CALIFORNIA SEA LION (ZALOPHUS CALIFORNIANUS) CARCINOMA-ASSOCIATED OTARINE HERPESVIRUS-1.

Otarine herpesvirus 1 (OtHV1) is strongly associated with California sea lion (CSL, Zalophus californianus) urogenital carcinoma, the most common cancer documented in marine mammals. In addition to CSL, OtHV1 has also been found in association with carcinoma in South American fur seals (Arctocephalus australis), demonstrating it can infect related species. Northern fur seals (NFS, Callorhinus ursinus) are sympatric with CSL, and copulation between these species has been observed; yet, there are no reports of urogenital carcinoma in NFS. We describe a new Otarine herpesvirus found in vaginal swabs from NFS, herein called OtHV4. Partial sequencing of the polymerase gene and the glycoprotein B gene revealed OtHV4 is closely related to OtHV1, with 95% homology in the region of polymerase sequenced, and phylogenetic analyses demonstrate that they are sister taxa. An OtHV4-specific hydrolysis probe quantitative PCR was developed and validated, and its use on vaginal swabs revealed 16 of 50 (32%) wild adult female NFS were positive for OtHV4. The identification of a virus highly similar to the carcinoma-associated OtHV1 in a sympatric species without carcinoma suggests that comparative genomics of OtHV1 and OtHV4 may identify candidate viral oncogenes.

Novel polyomaviral infection in the placenta of a northern fur seal (Callorhinus ursinus) on the Pribilof Islands, Alaska, USA.

Viruses of the family Polyomaviridae infect a wide variety of avian and mammalian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. In 2010, intranuclear viral inclusion bodies were identified in trophoblasts of a single northern fur seal (NFS; Callorhinus ursinus) placenta from a presumed healthy birth on St. Paul Island, Alaska. On transmission electron microscopy, virions were approximately 40 nm in diameter and were arranged in paracrystalline arrays within the nucleus. The tissue was positive for the polyomaviral major capsid gene (VP1) by PCR, and the sequenced product revealed a novel Orthopolyomavirus. Twenty-nine additional NFS placentas, devoid of viral inclusions on histologic examination, were tested for polyomavirus by PCR; all were negative. The significance of this novel virus for the infected animal is unknown, but the virus does not appear to be very prevalent within the placentas from newborn northern fur seal pups.

Serologic survey for potential pathogens and assessment of disease risk in Australian fur seals.

The introduction of pathogens into populations of animals with no previous exposure to them and, therefore, no immunologic protection, can result in epizootics. Predicting the susceptibility of populations to infectious diseases is crucial for their conservation and management. Australian fur seals (Arctocephalus pusillus doriferus) have a relatively small population size, a restricted range, and form dense aggregations. These factors make this species vulnerable to epizootics of infectious diseases that spread by direct animal-to-animal contact. Blood samples were collected from 125 adult female Australian fur seals between 2007 and 2009 and tested for exposure to selected pathogens. The testing protocol was based on pathogens important to marine mammal health or those significant to public and livestock health. No antibodies were detected to morbilliviruses, influenza A viruses, six Leptospira serovars, Mycobacterium tuberculosis-complex species, or Toxoplasma gondii. Overall antibody prevalence to an unidentified Brucella sp. was 57% but varied significantly (P<0.02) between 2007 (74%) and 2008 (53%). The findings indicate Brucella infection may be enzootic in the Australian fur seal population. Further investigations are required to isolate the bacteria and establish if infection results in morbidity and mortality. Australian fur seals remain vulnerable to the threat of introduced disease and should be managed and monitored accordingly.

Rotavirus infections in Galapagos sea lions.

Group A rotaviruses infect and cause diarrhea in the young of a broad range of terrestrial mammals, but it is unknown, to our knowledge, whether they infect marine mammals. During February and March of 2002 and 2003, we collected 125 serum samples and 18 rectal swab samples from Galapagos sea lion pups (GSL, Zalophus wollebaeki), and 22 serum samples from Galapagos fur seal pups (GFS, Arctocephalus galapagoensis) from nine islands of the Galapagos archipelago, Ecuador. Sera were tested for antibodies (immunoglobulin G [IgG]) to rotavirus by an enzyme immunoassay using rhesus rotavirus as the capture antigen. In addition, rectal swabs were analyzed for the presence of rotavirus genomic double-stranded RNA by silver-stained polyacrylamide gel electrophoresis. Antibodies to rotavirus were detected in 27 GSL pups (22%) and five GFS pups (23%), and rotavirus RNA was detected in the fecal sample from one GSL pup (6%). These results provide the first evidence that rotavirus infections are prevalent at an early age in Galapagos sea lions and Galapagos fur seals.

Serologic evidence of influenza A and B viruses in South American fur seals (Arctocephalus australis).

The aim of this work was to detect serologic evidence of influenza virus infections in South American fur seals (Arctocephalus australis) that inhabit the Uruguayan coast. In 29 of 37 serum samples that were analyzed, we identified antibodies to at least one of the following antigens: H1N1 (A/NewCaledonia/20/99), B/Beijing/184/93-like viruses, B/Hong Kong/330/01, and B/Sichuan/379/99 by means of the hemagglutination inhibition assay (HAI). Results confirmed that influenza A viruses circulate in marine mammals and also showed, for the first time, indirect evidence of influenza B infections in Arctocephalus australis.