Coronaviruses Associated with the Superfamily Musteloidea.

Among the animal superfamily Musteloidea, which includes those commonly known as mustelids, naturally occurring and species-specific alphacoronavirus infections have been observed in both mink (Mustela vison/Neovison vison) and domestic ferrets (Mustela putorius furo). Ferret systemic coronavirus (FRSCV), in particular, has been associated with a rare but fatal systemic disease. In recent months, it has become apparent that both minks and ferrets are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus and the cause of the coronavirus disease 2019 (COVID-19) pandemic. Several mink farms have experienced SARS-CoV-2 outbreaks, and experimental models have demonstrated susceptibility of ferrets to SARS-CoV-2. The potential for pet ferrets to become infected with SARS-CoV-2, however, remains elusive. During the 2002-2003 SARS epidemic, it was also apparent that ferrets were susceptible to SARS-CoV and could be utilized in vaccine development. From a comparative standpoint, understanding the relationships between different infections and disease pathogenesis in the animal superfamily Musteloidea may help elucidate viral infection and transmission mechanisms, as well as treatment and prevention strategies for coronaviruses.

Marine Morbilliviruses: Diversity and Interaction with Signaling Lymphocyte Activation Molecules.

Epidemiological reports of phocine distemper virus (PDV) and cetacean morbillivirus (CeMV) have accumulated since their discovery nearly 30 years ago. In this review, we focus on the interaction between these marine morbilliviruses and their major cellular receptor, the signaling lymphocyte activation molecule (SLAM). The three-dimensional crystal structure and homology models of SLAMs have demonstrated that 35 residues are important for binding to the morbillivirus hemagglutinin (H) protein and contribute to viral tropism. These 35 residues are essentially conserved among pinnipeds and highly conserved among the Caniformia, suggesting that PDV can infect these animals, but are less conserved among cetaceans. Because CeMV can infect various cetacean species, including toothed and baleen whales, the CeMV-H protein is postulated to have broader specificity to accommodate more divergent SLAM interfaces and may enable the virus to infect seals. In silico analysis of viral H protein and SLAM indicates that each residue of the H protein interacts with multiple residues of SLAM and vice versa. The integration of epidemiological, virological, structural, and computational studies should provide deeper insight into host specificity and switching of marine morbilliviruses.

Surveillance for highly pathogenic influenza A viruses in California during 2014-2015 provides insights into viral evolutionary pathways and the spatiotemporal extent of viruses in the Pacific Americas Flyway.

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014-2015 to (i) evaluate IAV prevalence in waterfowl, (ii) assess the evidence for spill-over infections in marine mammals and (iii) genetically characterize low-pathogenic (LP) and HP IAVs to refine inference on the spatiotemporal extent of HP genome constellations and to evaluate possible evolutionary pathways. We screened samples from 1496 waterfowl and 1142 marine mammals collected from April 2014 to August 2015 and detected IAV RNA in 159 samples collected from birds (n=157) and pinnipeds (n=2). HP IAV RNA was identified in three samples originating from American wigeon (Anas americana). Genetic sequence data were generated for a clade 2.3.4.4 HP IAV-positive diagnostic sample and 57 LP IAV isolates. Phylogenetic analyses revealed that the HP IAV was a reassortant H5N8 virus with gene segments closely related to LP IAVs detected in mallards (Anas platyrhynchos) sampled in California and other IAVs detected in wild birds sampled within the Pacific Americas Flyway. In addition, our analysis provided support for common ancestry between LP IAVs recovered from waterfowl sampled in California and gene segments of reassortant HP H5N1 IAVs detected in British Columbia, Canada and Washington, USA. Our investigation provides evidence that waterfowl are likely to have played a role in the evolution of reassortant HP IAVs in the Pacific Americas Flyway during 2014-2015, whereas we did not find support for spill-over infections in potential pinniped hosts.

Influenza Virus Infection of Marine Mammals.

Interspecies transmission may play a key role in the evolution and ecology of influenza A viruses. The importance of marine mammals as hosts or carriers of potential zoonotic pathogens such as highly pathogenic H5 and H7 influenza viruses is not well understood. The fact that influenza viruses are some of the few zoonotic pathogens known to have caused infection in marine mammals, evidence for direct transmission of influenza A virus H7N7 subtype from seals to man, transmission of pandemic H1N1 influenza viruses to seals and also limited evidence for long-term persistence of influenza B viruses in seal populations without significant genetic change, makes monitoring of influenza viruses in marine mammal populations worth being performed. In addition, such monitoring studies could be a great tool to better understand the ecology of influenza viruses in nature.

Phocine distemper virus: current knowledge and future directions.

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors.

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid--α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

Sealpox virus in marine mammal rehabilitation facilities, North America, 2007-2009.

Sealpox, a zoonotic disease affecting pinnipeds (seals and sea lions), can occur among captive and convalescing animals. We surveyed 1 worker each from 11 marine mammal centers and interviewed 31 other marine mammal workers to ascertain their knowledge of and experience with sealpox virus and to identify factors associated with sealpox virus outbreaks among pinnipeds in marine rehabilitation facilities. Demographic and health data were obtained for 1,423 pinnipeds at the 11 facilities. Among the 23 animals in which sealpox was clinically diagnosed, 4 arrived at the facility ill, 11 became ill <5 weeks after arrival, and 2 became ill ≥5 weeks after arrival; the timing of illness onset was unknown for 6 animals. Most infections occurred in pinnipeds <1 year of age. Nine affected animals were malnourished; 4 had additional illnesses. Sealpox had also occurred among workers at 2 facilities. Sealpox is a noteworthy zoonosis of rehabilitating convalescing pinnipeds; workplace education can help to minimize risks for human infection.

Phylogenetic analysis of marine mammal herpesviruses.

Five novel DNA-dependent DNA polymerase (Dpol) herpesviral sequences were generated using nested consensus polymerase chain reaction (PCR) in clinical samples from a harbor seal (Phoca vitulina), bottlenose dolphin (Tursiops truncatus), orca (Orcinus orca), California sea lion (Zalophus californianus), and a Phocid herpesvirus 2 (PhHV-2) isolate from a harbor seal (used as positive control). These novel sequences and other representative herpesvirus sequences were included in Bayesian and Maximum Likelihood analyses to illustrate the phylogeny of herpesviruses amongst the marine mammal host species and in comparison to those of other animals. All 19 novel and known marine mammal herpesviruses included in the analyses aligned with members of the Alphaherpesvirinae or Gammaherpesvirinae subfamilies. The novel harbor seal herpesvirus clustered with members of the Macavirus genus, subfamily Gammaherpesvirinae. The novel bottlenose dolphin herpesvirus clustered together in a monophyletic group with another delphinid alphaherpesvirus but could not be associated with an established genus. The orca herpesvirus also clustered with a delphinid alphaherpesvirus and formed a separate clade. The sea lion herpesvirus clustered with PhHV-2. PhHV-1 clustered with varicelloviruses and PhHV-2 clustered strongly in the Gammaherpesvirinae genus Percavirus. All cetacean gammaherpesviruses formed a monophyletic clade and could not be associated with an established gammaherpesviral genus.

Genetic identification of novel poxviruses of cetaceans and pinnipeds.

Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

Morbillivirus infections in aquatic mammals: a brief overview.

Since 1987, at least eight morbillivirus infection (MI) epidemics have caused mass mortality of several free-living pinniped and cetacean populations around the world. The responsible agents, all belonging to the genus Morbillivirus (family Paramyxoviridae), have been characterized as either "canine distemper virus" strains, infecting pinnipeds, or as three new morbilliviruses, namely "phocid (phocine) distemper virus" , "porpoise morbillivirus" and "dolphin morbillivirus" . The last two agents are currently gathered under the common denomination of "cetacean morbillivirus". At post-mortem examination, a commonly occurring macroscopic lesion is represented by more or less severe bilateral pneumonia, with consolidation, congestion and oedema of both lungs, which fail to collapse. Histologically, a non-suppurative broncho-interstitial pneumonia, characterized by type II pneumocyte hyperplasia and by formation of endobronchial, endobronchiolar and endoalveolar "Warthin-Finkeldey type" syncytia, as well as a multifocal, non-suppurative encephalitis, associated with a severe and generalized lymphoid tissue depletion, are common pathological findings. Furthermore, eosinophilic viral inclusions are often detected, at both the intracytoplasmic and intranuclear level, within bronchial and bronchiolar epithelial, pulmonary syncytial, neuronal and other cell types. These inclusions, along with lymphoid and other cellular elements, are often found to be immunohistochemically positive for morbillivirus antigen. Among the still debated, or even controversial issues regarding MI in sea mammals, the one related to the origin of their causative agents is of particular concern. Another intriguing issue regards the synergistic effects, if any, associated with chronic exposure to a number of environmental pollutants, such as organochlorines and heavy metals. In fact, it is also unknown whether and how these chemicals contribute towards modulating the pathogenic and pathogenetic activity primarily displayed by sea mammal morbilliviruses.

Emerging morbillivirus infections of marine mammals: development of two diagnostic approaches.

In the last 13 years, four viruses belonging in the Morbillivirus genus of the Paramyxoviridae family have emerged as significant causes of disease and mortality in marine mammals. The viruses involved are canine distemper virus (CDV) in seals and polar bears, dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) in cetaceans, and phocine distemper virus (PDV) in pinnipeds. The two cetacean morbilliviruses (DMV and PMV) are now considered to be the same viral species, named cetacean morbillivirus (CMV). All three morbillivirus species (CDV, CMV, and PDV) are genetically and antigenically related and cross-react in various serological tests. The diagnosis of morbilliviral infections in marine mammal specimens poses two challenges. First, various marine mammal species can be infected by more than one closely related but distinct morbilliviruses, making definitive virus identification unattainable by classical virology methods. Second, standard immunological reagents such as anti-species conjugates are unavailable for most marine mammal species, rendering definitive serological diagnosis difficult by classical serological techniques. The objectives of this study were to develop two diagnostic approaches that alleviate these difficulties, providing simple, rapid, and cost-effective diagnostic methods. For nucleic acid detection, reverse transcription-polymerase chain reaction (RT-PCR) and restriction endonuclease digestions were used to differentiate the three viruses. For antibody detection, a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (c-ELISA) was used on sera from several species, thus avoiding the need for multiple anti-species enzyme conjugates.

Characterization of phocid herpesvirus-1 and -2 as putative alpha- and gammaherpesviruses of North American and European pinnipeds.

To study the relationships between herpesvirus recently isolated from different pinniped species, antigenic and genetic analyses were performed. First, herpesviruses isolated from North American harbour seals (Phoca vitulina), a Californian sea lion (Zalophus californianus) and a European grey seal (Halichoerus grypus) were examined in an enzyme immunoassay (EIA) with a panel of monoclonal antibodies which had previously been shown to allow typing of herpesviruses from European harbour seals into two distinct virus types: phocid herpesvirus type-1 and type-2 (PhHV-1 and PhHV-2). The EIA data showed that all but one of the isolates from seals ranging in United States coastal waters were PhHV-2-like while the European grey seal herpesvirus was PhHV-1-like. Genetic characterization was facilitated by PCR analysis using primers based on conserved regions of the glycoprotein B and D (gB and gD) genes of the antigenically closely related canid (CHV) and felid (FHV) herpesvirus. Specific amplified products were obtained with five isolates antigenically characterized as PhHV-1-like but not with five PhHV-2-like isolates. Sequence analysis of the PCR products confirmed greatest similarity to members of the genus Varicellovirus of the Alphaherpesvirinae and in particular to CHV. Sequence analysis of two EcoRI fragments of the PhHV-2 genome (European isolate 7848) revealed greatest similarity to gammaherpesviruses and in particular equine herpesvirus-2. Although an unambiguous subgrouping was not feasible, this is the first evidence that PhHV-2 may be a putative gammaherpesvirus of pinnipeds.