In the Search of Marine Pestiviruses: First Case of Phocoena Pestivirus in a Belt Sea Harbour Porpoise.

Pestiviruses are widespread pathogens causing severe acute and chronic diseases among terrestrial mammals. Recently, Phocoena pestivirus (PhoPeV) was described in harbour porpoises (Phocoena phocoena) of the North Sea, expanding the host range to marine mammals. While the role of the virus is unknown, intrauterine infections with the most closely related pestiviruses- Bungowannah pestivirus (BuPV) and Linda virus (LindaV)-can cause increased rates of abortions and deaths in young piglets. Such diseases could severely impact already vulnerable harbour porpoise populations. Here, we investigated the presence of PhoPeV in 77 harbour porpoises, 277 harbour seals (Phoca vitulina), grey seals (Halichoerus grypus) and ringed seals (Pusa hispida) collected in the Baltic Sea region between 2002 and 2019. The full genome sequence of a pestivirus was obtained from a juvenile female porpoise collected along the coast of Zealand in Denmark in 2011. The comparative Bayesian phylogenetic analyses revealed a close relationship between the new PhoPeV sequence and previously published North Sea sequences with a recent divergence from genotype 1 sequences between 2005 and 2009. Our findings provide further insight into the circulation of PhoPeV and expand the distribution from the North Sea to the Baltic Sea region with possible implications for the vulnerable Belt Sea and endangered Baltic Proper harbour porpoise populations.

Phocine distemper virus uses phocine and other animal SLAMs as a receptor but not human SLAM.

Morbilliviruses use the signaling lymphocyte activation molecule (SLAM) as a receptor to infect their hosts. Seals are almost the only animal species that show apparent infection with phocine distemper virus (PDV). Seal SLAM functioned as a PDV receptor. However, dolphin- and dog-SLAM molecules, but not human SLAM, were also fully functional PDV receptors. These data suggest that the host range of PDV is not simply determined by its SLAM usage. However, human nonsusceptibility to PDV infection may be at least partly attributable to the inability of PDV to use human SLAM as a receptor.

BACTERIAL MICROBIOTA IN HARBOR SEALS (PHOCA VITULINA) FROM THE NORTH SEA OF SCHLESWIG-HOLSTEIN, GERMANY, AROUND THE TIME OF MORBILLIVIRUS AND INFLUENZA EPIDEMICS.

We present microbiologic findings in harbor seal (phoca; Phoca vitulina ) carcasses collected from the North Sea of Schleswig-Holstein, Germany, 1996-2014, and interpret results in relation to potential variations caused by phocine distemper virus and influenza A virus mass mortalities. We conducted microbiologic investigations on 2,124 tissue samples from lung, liver, kidney, spleen, intestine, and mesenteric lymph nodes from 549 dead harbor seals of the German North Sea. A large variety of bacteria, including potentially pathogenic species such as Bordetella bronchiseptica , Brucella spp., Clostridium perfringens , Escherichia coli , Erysipelothrix rhusiopathiae , ß-hemolytic streptococci, and Staphylococcus aureus , were isolated. These bacteria were associated with bronchopneumonia, gastroenteritis, hepatitis, polyarthritis, nephritis, myositis, myocarditis, and septicemia. Bordetella bronchiseptica and Streptococcus equi subsp. zooepidemicus were significantly associated with the seal die-offs from phocine distemper in 2002 and influenza in 2014. Many bacteria were detected in tissues of dead harbor seals, of which E. coli , ß-hemolytic streptococci, and Brucella spp. might be responsible for pathologic changes. Zoonotic bacteria such as Brucella spp. and E. rhusiopathiae are frequently isolated from harbor seals. Brucella spp. was less and Vibrio spp. more frequently found in summer. Erysipelothrix rhusiopathiae showed an almost regular 4-yr oscillating trend. We found C. perfringens less frequently and E. coli more frequently in harbor seals from St. Peter-Ording. Because zoonotic bacteria are regularly found, handling of dead and live harbor seal specimens should be conducted carefully to prevent transmission to humans. Further investigations are needed to understand microbiota changes in relation to increasing seal populations, reintroduction of rehabilitated seals to the wild, and increasing pressure from anthropogenic activities.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.

Phocine distemper virus (PDV) seroprevalence as predictor for future outbreaks in harbour seals.

Phocine distemper virus (PDV) infections caused the two most pronounced mass mortalities in marine mammals documented in the past century. During the two outbreaks, 23,000 and 30,000 harbour seals (Phoca vitulina), died in 1988/1989 and 2002 across populations in the Wadden Sea and adjacent waters, respectively. To follow the mechanism and development of disease spreading, the dynamics of Morbillivirus-specific antibodies in harbour seal populations in German and Danish waters were examined. 522 serum samples of free-ranging harbour seals of different ages were sampled between 1990 and 2014. By standard neutralisation assays, Morbillivirus-specific antibodies were detected, using either the PDV isolate 2558/Han 88 or the related canine distemper virus (CDV) strain Onderstepoort. A total of 159 (30.5%) of the harbour seals were seropositive. Annual seroprevalence rates showed an undulating course: Peaks were seen in the post-epidemic years 1990/1991 and 2002/2003. Following each PDV outbreak, seroprevalence decreased and six to eight years after the epidemics samples were tested seronegative, indicating that the populations are now again susceptible to new PDV outbreak. After the last outbreak in 2002, the populations grew steadily to an estimated maximum (since 1975) of about 39,100 individuals in the Wadden Sea in 2014 and about 23,540 harbour seals in the Kattegat area in 2013. A re-appearence of PDV would presumably result in another epizootic with high mortality rates as encountered in the previous outbreaks. The current high population density renders harbour seals vulnerable to rapid spread of infectious agents including PDV and the recently detected influenza A virus.

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe.

UNLABELLED: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host. IMPORTANCE: Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.

Seroprevalence of Antibodies against Seal Influenza A(H10N7) Virus in Harbor Seals and Gray Seals from the Netherlands.

In the spring and summer 2014, an outbreak of seal influenza A(H10N7) virus infection occurred among harbor seals (Phoca vitulina) off the coasts of Sweden and Denmark. This virus subsequently spread to harbor seals off the coasts of Germany and the Netherlands. While thousands of seals were reported dead in Sweden, Denmark and Germany, only a limited number of seals were found dead in the Netherlands. To determine the extent of exposure of seals in the Netherlands to influenza A/H10N7 virus, we measured specific antibody titers in serum samples from live-captured seals and seals admitted for rehabilitation in the Netherlands by use of a hemagglutination inhibition assay and an ELISA. In harbor seals in 2015, antibodies against seal influenza A(H10N7) virus were detected in 41% (32 out of 78) pups, 10% (5 out of 52) weaners, and 58% (7 out of 12) subadults or adults. In gray seals (Halichoerus grypus) in 2015, specific antibodies were not found in the pups (n = 26), but in 26% (5 out of 19) of the older animals. These findings indicate that, despite apparent low mortality, infection with seal influenza A(H10N7) virus was geographically widespread and also occurred in grey seals.

Brain Meta-Transcriptomics from Harbor Seals to Infer the Role of the Microbiome and Virome in a Stranding Event.

Marine diseases are becoming more frequent, and tools for identifying pathogens and disease reservoirs are needed to help prevent and mitigate epizootics. Meta-transcriptomics provides insights into disease etiology by cataloguing and comparing sequences from suspected pathogens. This method is a powerful approach to simultaneously evaluate both the viral and bacterial communities, but few studies have applied this technique in marine systems. In 2009 seven harbor seals, Phoca vitulina, stranded along the California coast from a similar brain disease of unknown cause of death (UCD). We evaluated the differences between the virome and microbiome of UCDs and harbor seals with known causes of death. Here we determined that UCD stranded animals had no viruses in their brain tissue. However, in the bacterial community, we identified Burkholderia and Coxiella burnetii as important pathogens associated with this stranding event. Burkholderia were 100% prevalent and ~2.8 log2 fold more abundant in the UCD animals. Further, while C. burnetii was found in only 35.7% of all samples, it was highly abundant (~94% of the total microbial community) in a single individual. In this harbor seal, C. burnetii showed high transcription rates of invading and translation genes, implicating it in the pathogenesis of this animal. Based on these data we propose that Burkholderia taxa and C. burnetii are potentially important opportunistic neurotropic pathogens in UCD stranded harbor seals.

Influenza A(H10N7) virus in dead harbor seals, Denmark.

Since April 2014, an outbreak of influenza in harbor seals has been ongoing in northern Europe. In Denmark during June-August, 152 harbor seals on the island of Anholt were found dead from severe pneumonia. We detected influenza A(H10N7) virus in 2 of 4 seals examined.

Identification of DNA sequences that imply a novel gammaherpesvirus in seals.

Various herpesviruses have been discovered in marine mammals and are associated with a wide spectrum of disease. In the present study we describe the detection and phylogenetic analysis of a novel gammaherpesvirus, tentatively called phocine herpesvirus 7 (PhHV-7), which was detected in samples collected during an outbreak of ulcerative gingivitis and glossitis from juvenile harbour seals (Phoca vitulina) at the Seal Rehabilitation and Research Centre, the Netherlands. The presence of this novel gammaherpesvirus was confirmed by viral metagenomics, while no other viruses other than four novel anelloviruses were detected. However, PhHV-7 DNA was also detected in harbour and grey seals (Halichoerus grypus) without gingivitis or glossitis. Genetic analysis of the partial polymerase gene of PhHV-7 detected in both species revealed limited sequence variation. Additional studies are needed to elucidate whether the viruses discovered played a role in the disease observed.

Structural and functional analysis of surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals.

UNLABELLED: In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (S. J. Anthony, J. A. St Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles, W. I. Lipkin, MBio 3:e00166-00112, 2012, http://dx.doi.org/10.1128/mBio.00166-12). Here, we present a detailed structural and biochemical analysis of the surface antigens of the virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission, our results indicate that the A(H3N8) virus currently poses a low risk to humans. IMPORTANCE: Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here, we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. The results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses as these zoonotic influenza viruses continue to circulate and adapt to new hosts.

Avian influenza A(H10N7) virus involvement in mass mortality of harbour seals (Phoca vitulina) in Sweden, March through October 2014.

We provide the first scientific report of influenza A virus involvement in a mass mortality event among harbour seals (Phoca vitulina) off the west coast of Sweden. Avian influenza A (H10N7) virus was detected in the lungs of two affected animals. This subtype has not been reported in seals to date, nor has influenza A-associated mortality been reported in seals in Europe. Circulation of avian influenza viruses in mammals may have implications for public health.

Molecular epidemiology of seal parvovirus, 1988-2014.

A novel parvovirus was discovered recently in the brain of a harbor seal (Phoca vitulina) with chronic meningo-encephalitis. Phylogenetic analysis of this virus indicated that it belongs to the genus Erythroparvovirus, to which also human parvovirus B19 belongs. In the present study, the prevalence, genetic diversity and clinical relevance of seal parvovirus (SePV) infections was evaluated in both harbor and grey seals (Halichoerus grypus) that lived in Northwestern European coastal waters from 1988 to 2014. To this end, serum and tissue samples collected from seals were tested for the presence of seal parvovirus DNA by real-time PCR and the sequences of the partial NS gene and the complete VP2 gene of positive samples were determined. Seal parvovirus DNA was detected in nine (8%) of the spleen tissues tested and in one (0.5%) of the serum samples tested, including samples collected from seals that died in 1988. Sequence analysis of the partial NS and complete VP2 genes of nine SePV revealed multiple sites with nucleotide substitutions but only one amino acid change in the VP2 gene. Estimated nucleotide substitution rates per year were 2.00 × 10(-4) for the partial NS gene and 1.15 × 10(-4) for the complete VP2 gene. Most samples containing SePV DNA were co-infected with phocine herpesvirus 1 or PDV, so no conclusions could be drawn about the clinical impact of SePV infection alone. The present study is one of the few in which the mutation rates of parvoviruses were evaluated over a period of more than 20 years, especially in a wildlife population, providing additional insights into the genetic diversity of parvoviruses.

Novel B19-like parvovirus in the brain of a harbor seal.

Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.

Phocine herpesvirus 1 (PhHV-1) in harbor seals from Svalbard, Norway.

Phocine herpesvirus 1 (PhHV-1) infections in seals are associated with disease and sometimes high mortality, primarily in young animals. PhHV-1 has been detected in seals from European waters as well as in waters on both coasts of North America. Serological surveys of various pinniped species have indicated a wide geographical distribution of PhHV-1. A quantitative and sensitive real-time PCR assay targeting the gene encoding glycoprotein B of PhHV-1 was developed for detection of PhHV-1 in ocular and nasal swab samples from wild harbor seals (Phoca vitulina) from Svalbard (Norway). PhHV-1 DNA was detected in samples from 6 (8%) seals collected in 2009 and 2 (3%) in 2010; all had herpesvirus DNA in the ocular swab sample, whereas only one of these animals also had herpesvirus DNA in the nasal swab sample. Four PCR positive animals were approximately 1 year of age and four were pups of the year. Serum samples obtained in 1998 (n=59), 1999 (n=74), 2000 (n=81), 2009 (n=69) and 2010 (n=83) were tested for anti-PhHV-1 antibodies in an indirect ELISA. The PhHV-1 seroprevalence in the population remained high throughout this period, varying from 77 to 100% between years. No eye disease was observed in this harbor seal population, but the ELISA and PCR findings reported here suggest that PhHV-1 is endemic in this globally northernmost harbor seal colony, and that the virus is shed on the mucosa of the eye and nose.

Dolphin morbillivirus infection in a captive harbor seal (Phoca vitulina).

During the second morbillivirus epidemic (2007 to 2011) in cetaceans along the Italian coastline, dolphin morbillivirus (DMV) was detected by molecular analyses in a captive harbor seal (Phoca vitulina), with pathological findings consistent with morbillivirus infection. This report confirms interspecies DMV transmission from cetaceans to pinnipeds.

Emergence of fatal avian influenza in New England harbor seals.

UNLABELLED: From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission. IMPORTANCE: The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.