Five animals were submitted for anatomical and microbiological analysis. All were collected from the peak of the outbreak (late September to October) and had pneumonia and ulcerations of the skin and oral mucosa (). Nucleic acids extracted from lung, trachea, liver, kidney, thoracic lymph node, mesenteric lymph node, spleen, skin lesion, and oral mucosa were tested by PCR for the presence of a wide range of pathogens, including herpesviruses, poxviruses, adenoviruses, polyomaviruses, caliciviruses, paramyxoviruses, astroviruses, enteroviruses, flaviviruses, rhabdoviruses, orbiviruses, and influenza viruses. Influenza A virus was detected in several tissues from all five animals, and PCR cloning and sequencing of genes for hemagglutinin (HA) and neuraminidase (NA) revealed the subtype to be H3N8, a subtype typically associated with infection of avian, equine, and canine hosts (6-8
). Influenza virus was isolated from the allantoic fluid of specific-pathogen-free (SPF) eggs inoculated with homogenates of PCR-positive tissues, including lung, lymph nodes, tonsil, and kidney, and all isolates were reconfirmed to be H3N8. In accord with conventional nomenclature, the virus is provisionally named A/harbor seal/Massachusetts/1/2011.
FIG 1 Hematoxylin and eosin staining of the lung at a ×10 magnification. There is diffuse acute interstitial pneumonia and a mix of acute hemorrhagic alveolitis with necrotizing bronchitis. Multifocally, alveoli are either filled with hemorrhage and (more ...) In situ
hybridization (ISH) using oligonucleotide probes for influenza virus H3N8 segments 4 (HA) and 7 (matrix) and immunohistochemistry using polyclonal antibodies against H3N8 HA antigen confirmed the presence of influenza virus in lung, where signal was concentrated in the bronchiole epithelium and mucosa of the pulmonary parenchyma ( and see Fig. S1
in the supplemental material). The average load of HA and NA RNAs in lung was 300 copies/100 ng of extracted RNA. ISH staining in nonrespiratory tissues was limited to sporadic infection of single cells in intestine, kidney, and lymph node, and the average HA/NA RNA load was five copies/100 ng. These results are consistent with the histological observation that the main site of viral replication is the respiratory tract. Cellular morphology and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) consistent with apoptosis was observed in virus-infected pulmonary epithelial cells (). TUNEL staining also revealed the widespread presence of apoptotic cells in areas where no virus was observed but not in negative-control tissue, suggesting an additional host-mediated response to the infection.
FIG 2 (A and B) Fluorescent in situ hybridization (ISH) of H3N8 virus-infected seal cells with DAPI counterstaining. A probe targeting the viral hemagglutinin demonstrates diffuse infection of the bronchial mucosal epithelium. ISH was also performed using probes (more ...)
FIG 3 (A) Immunohistochemistry (IHC) of seal bronchus. Polyclonal antibodies were raised against H3N8 virus-specific HA antigen. Brown staining (DAB reporter system) indicates the presence of viral antigen. There is irregularity of the mucosal surface, with (more ...)
Full-genome sequencing was completed following PCR cloning of all eight influenza genome segments directly from infected tissue. Sequences were submitted to GenBank and assigned accession numbers JQ433879 to JQ433882. Phylogenetic analyses of these nucleotide sequences with avian, canine, and equine H3N8 influenza virus genomes demonstrated the closest relationship to a virus identified in North American waterfowl in all 8 segments (). These data are consistent with the recent transmission of the H3N8 virus from wild birds to seals. The closest avian relative, A/blue-winged teal/Ohio/926/2002, had an overall 96.07% nucleotide sequence identity across the genome (Hamming distance), with no individual segment having less than 94% identity. This level of similarity across all segments with an isolate separated by a span of 10 years suggests that this virus has been circulating in the aquatic bird populations since at least 2002.
FIG 4 Phylogenetic trees of representative influenza H3N8 genome segments. Nucleotide sequence alignments for all genome segments were created using ClustalW, and trees were produced using neighbor-joining, maximum-likelihood, and Bayesian algorithms. Models (more ...)
A total of 37 amino acid substitutions separate the seal H3N8 and avian H3N8 viruses, which are summarized in . The corresponding amino acids found in other seal influenza viruses, in the canine and equine H3N8 viruses, and in selected human influenza viruses are included for comparison. Of these, mutations PB2-701N and HA-260M are shared exclusively by the seal and mammalian (canine/equine) H3N8 viruses, while mutations NA-399R, PB2-382V, and PA-184N are shared by the seal H3N8 and human H3N2 viruses, all of which suggests adaptation to mammalian hosts. Mutations PB2-60N, PB2-376R, PB1-174I, PB1-309G, PB1-359G, PB1-376V, PB1-377G, PB1-464E, NP-63V, NP-128G, and NP-296H are all exclusively found in the seal H3N8 virus (). Future studies will be required to assess the functional significance of many of these mutations, especially in seal H3N8 PB1, where a significant number of exclusive mutations were observed. Given the importance of PB1 in viral replication, it is probable that these mutations represent adaptive selection to accommodate host-specific differences in intracellular replication.
TABLE 1 List of amino acid substitutions between seal H3N8 and avian H3N8 virusesa
The seal H3N8 genome was interrogated for any genetic features that might contribute to enhanced transmissibility and virulence in seals. Expression of a second peptide (PB1-F2) from segment 2 has been associated with an increase in pathogenicity by inducing apoptosis and increasing both inflammation and secondary bacterial pneumonia (9
). The seal H3N8 virus contains an intact open reading frame for the pathogenic version of this accessory protein, which includes a serine at amino acid position 66 (9
). All five seals had evidence of apoptosis and secondary bacterial pneumonia.
Glycosylation can also affect pathogenicity in influenza viruses (11-14
). Six potential glycosylation sites were detected in the seal H3N8 HA, based on the sequence X−2
, at amino acid positions 24, 38, 54, 181, 301, and 499. None had features suggestive of inactivity or reduced efficiency, as was previously demonstrated for H5N2 (12
). Many H5N1 viruses have an additional glycosylation site at positions 158 to 160, and previous work demonstrated that the deletion of this site is critical for H5N1 viruses to bind to human-SAα2,6-like receptors and to transmit between mammals (15
). This glycosylation site is missing in the seal H3N8 HA.
In order to investigate the specificity of sialic acid binding, the seal virus was compared to avian H5 and swine H9, both of which bind to the sialyloligosaccharide SAα2,3 ligand in a structural-homology model (). All structures confirmed the presence of a highly conserved serine at position 152 (corresponding to S136 by H3 numbering [16, 17]), which lies in a binding pocket, where its hydroxyl group contacts the axial carboxylate of sialic acid (17
). While the seal virus contains the same conserved S152, it also harbors a neighboring G151E mutation (), which introduces a large residue capable of both donating and receiving hydrogen bonds with residues in close proximity to the ligand-binding pocket. Rotamer hydrogen bond analysis of the modeled seal structure indicates that HA’s altered conformation results in reduced hydrogen bonding between the conserved serine and SAα2,3 compared to that of H5 and H9 influenza viruses. Such changes in sialic acid binding play important roles in novel host adaptation (18
FIG 5 Structural-homology model showing the interaction of influenza HA with the SAα2,3 ligand. Seal H3 (gray), avian H5 (orange) (Protein Data Bank [PDB] accession number 1JSO), and swine H9 (pink) (PDB accession number 1JSH) were compared. The mutation (more ...)
Mutations at positions 226 and 228 (H3 numbering) in the H3 HA can also affect receptor-binding preferences and can either completely abrogate (Q226L) or reduce (G228S) affinity for the avian-preferred SAα-2,3 interaction (18
). Seal H3N8 virus maintains the avian phenotype at positions 226 (Q) and 228 (G), which correlates with a continued ability to use SAα-2,3. Together, these findings suggest that the seal virus may still be able to use SAα2,3, but perhaps with less efficiency than in its original avian host. Given this, we investigated whether seal H3N8 may have adapted and acquired an additional or increased affinity for the SAα-2,6 receptors that are more prevalent in mammalian respiratory tissue (20-23
The pulmonary distribution of SAα-2,3 and SAα-2,6 influenza receptors was investigated using the receptor-specific lectins Sambucus nigra
agglutinin (SNA) for SAα-2,6 and Maackia amurensis
lectin II (MAL II) for SAα-2,3. SAα-2,6 was widely expressed in both infected and noninfected pulmonary parenchyma, with the highest concentration seen on endothelial cells, followed by alveolar/bronchiole epithelia ( and see Fig. S2
in the supplemental material). SAα-2,3 was also observed, but less frequently, and was generally limited to the luminal surfaces of epithelial cells of the bronchioles. This broadly agrees with the expression of these SA saccharides in humans and pigs (20-23
) and demonstrates that seals do express receptors that would allow avian viruses to initiate infection. This observation is supported by the H3N3 seal virus from the 1991 epizootic (4
), which was shown to preferentially bind SAα-2,3 in vitro
). However, the limited prevalence of SAα-2,3 in the lower lung suggests that the process of infection is inefficient and may help to explain why epizootics of avian influenza occur but are infrequent in harbor seals.
Importantly, the rare expression of SAα-2,3 is insufficient to explain the diffuse infection seen throughout the pulmonary parenchyma (). In contrast, the wide distribution of SAα-2,6 is far more consistent with the level of infection observed. Costaining of infected lung with viral HA and SAα-2,3 or SAα-2,6 demonstrated clear infection of SAα-2,6-positive cells, in which no SAα-2,3 was seen ( and S3).
Hemagglutination assays were also performed to confirm sialic acid binding preferences. Seal H3N8 isolates were first sequenced to confirm that passage in eggs had not altered the HA phenotype detected in the infected tissues, and the viruses were then tested for their ability to agglutinate erythrocytes that preferentially express SAα-2,3 (horse) or SAα-2,6 (guinea pig, pig) (24
), relative to several avian H3N8 viruses (). Average agglutination titers for seal H3N8 virus with horse erythrocytes (1:48) show that the virus can still bind to SAα-2,3. However, titers were appreciably higher with guinea pig (1:192) and pig (1:144) erythrocytes, demonstrating a preference for SAα-2,6. These findings show that seal H3N8 can use both avian and mammalian receptors and add to previous studies that have demonstrated changes in receptor preferences following a host switch event (26
). The patterns of SAα-2,3 and SAα-2,6 binding to seal H3N8 virus also agree with the patterns observed for H3 avian viruses adapting to humans (18
FIG 6 Hemagglutination assays were performed on an isolate of the H3N8 seal virus (A/harbor seal/New Hampshire/179629/2011) to confirm sialic acid binding preferences. Viruses were tested for their ability to agglutinate erythrocytes that preferentially express (more ...)
A further mutation was observed in HA, this time at position 110 (). In avian H3 viruses, phenylalanine (Phe/F) is consistently seen, while seal H3N8 uses Ser (F110S). The significance of this (if any) is currently unknown; however, previous work has suggested that this amino acid (position 110) is a critical component of the influenza fusion peptide (27
), and given the essential role of fusion in viral replication and the host-specific differences that presumably exist in this process, the F110S substitution may well represent further adaption of this virus to mammalian replication.
The ability of avian influenza viruses to adapt to SAα-2,6-mediated cell entry and replication is regarded as a significant driving force in the emergence of global pandemics (19
), especially for viruses with phenotypes that confer increased virulence. Such phenotypes are often, though not exclusively, dictated by mutations in segment 1 (PB2), which is an important determinant of host range for influenza viruses. Previous studies have experimentally demonstrated the effect of various PB2 substitutions on virulence and transmissibility in mammalian hosts (15
), including the modification of the aspartic acid (D) avian phenotype to an asparagine (N) mammalian phenotype at amino acid 701 (15
). This D701N mutation has been experimentally introduced into an adapted version of the H7N7 seal influenza virus isolated in 1982 (1, 2) and was shown to increase the pathogenicity of the virus to mice (32
The seal H3N8 virus from the 2011 outbreak has naturally acquired this D701N substitution (), which was confirmed by clonal sequencing directly from infected tissue (50 clones/animal) to be the only phenotype present in all five animals. None of the previous outbreaks of influenza in seals showed this 701N phenotype, but it is consistently found in H3N8 viruses from horses and dogs, demonstrating further adaptation to replication in mammalian hosts. These observations raise significant concern about the virulence and transmission of this virus between mammals. Interestingly, analysis of HA sequences over the course of the outbreak show the introduction and maintenance of two nucleotide polymorphisms (), and while this is insufficient to convincingly demonstrate seal-to-seal transmission, it leads us to postulate that mammalian spread might already have occurred.
TABLE 2 Sequence analysis of the HA genes isolated from various tissuesa
Together, the adaptations observed in A/harbor seal/Massachusetts/1/11 suggest that it may be able to persist within the seal population and evolve into a new clade within the H3N8 group, as happened with the canine and equine viruses. An additional concern is the potential zoonotic threat that this virus poses, as it has already acquired mutations in both PB2 and HA that are often, though perhaps not exclusively, regarded as prerequisites for pandemic spread (19-23
) and it is uncertain how any persistence of the virus in mammals may continue to alter its phenotype. A comparison of A/harbor seal/Massachusetts/1/11 with human H3N2 viruses revealed three substitutions that are already common to both seal H3N8 and human H3N2 viruses. These are NA-W399R, PB2-I382V, and PA-S184N (). In all cases, these substitutions are shared by the seal H3N8 and human H3N2 viruses but are not found in influenza viruses isolated previously from seals, in avian, equine, or canine H3N8 viruses, or in either seasonal or pandemic H1N1 viruses. Further studies will be required to establish the functional significance of these substitutions; however, the natural epizootic emergence at this time of a pathogenic virus that can transmit between mammals, found in a species that can become infected with multiple influenza virus subtypes, must be considered a significant threat to both wildlife and public health.