Rapid communications Avian influenza A ( H 10 N 7 ) virus involvement in mass mortality of harbour seals ( Phoca vitulina ) in Sweden , March through October 2014

S Zohari (siamak.zohari@sva.se)1, A Neimanis2, T Härkönen3, C Moraeus3, J F Valarcher1 1. Department of Virology, Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden 2. Department of Pathology and Wildlife Diseases, Wildlife Section, National Veterinary Institute, Uppsala, Sweden 3. Department of Environmental Research and Monitoring, Swedish Museum of Natural History, Stockholm, Sweden


Background
Increased numbers of dead harbour seals (Phoca vitulina) from the west coast of Sweden were first noted in March 2014.From March through October, 425 carcasses were detected in several seal colonies in the Kattegat and the Skagerrak seas (Figure 1).This unusually high mortality contrasted with the typical annual number of 30 to 40 dead seals reported from this area.Although most seals were too decomposed for examination, influenza A virus (IAV) subtype H10N7

Figure 1
Cumulative numbers of dead stranded harbour seals (Phoca vitulina) along the west coast of Sweden, March-October 2014 (n=425) The arrows indicate the dates of sampling of the inuenza A positive seals.was detected in the lungs of two animals.According to media reports [1], H10N7 virus has recently been detected in dead seals in Denmark, Germany and the Netherlands in association with die-offs of seals first observed in July in Denmark and currently ongoing in Germany and the Netherlands [2,3].

Case descriptions
Seal 1 was observed suffering from buoyancy problems and respiratory distress off the coast of Gate Klova, Halland province.It was euthanised on 16 April, 2014 and examined by necropsy at the Swedish Museum of Natural History.Fresh and formalin-fixed tissues were submitted to the Swedish National Veterinary Institute (SVA) for further examination.Seal 2 was found dead on 29 August, 2014 in Beateberg, Västra Götaland

Figure 2 Phylogenetic relationship between haemagglutinin genes of H10 of influenza A virus subtypes
The protein coding region tree was generated by neighbour-joining analysis with the Tamura-Nei γ-model, using MEGA

Pathological findings
Formalin-fixed tissues from both seals were processed by routine histological examination [4].Seal 1 was a 12 year-old adult male in slightly impaired nutritional condition.Widespread emphysema was observed within the mediastinum and thoracic musculature, and bronchial and mediastinal lymph nodes were enlarged and oedematous.Lungs were diffusely firmer than normal, consistent with interstitial pneumonia, and the spleen was enlarged.Severe acute necrosuppurative pneumonia with widespread effacement of Seal 2 was severely decomposed and only a small sample of lung was available for examination.Despite the loss of cellular detail from autolysis, alveolar wall thickening supportive of an interstitial pneumonia was detected microscopically.Autolysis and limited material precluded investigation of concurrent bacterial pneumonia.

Virology
RNA was extracted from lung tissues and tested initially by real-time reverse-transcription polymerase chain reaction (rRT-PCR) targeting the matrix protein gene of avian influenza A viruses (AIV) [5] and the haemagglutinin gene of phocine distemper virus (PDV) [6].AIV RNA was detected in lung tissue of both Seals 1 and 2, while PDV was not detected.RT-PCR for detection of the haemagglutinin (HA) and neuraminidase (NA) genes of IAV was performed using segment-specific but subtype-universal primers as previously described [7].The nucleotide sequence A/ Seal/Sweden/SVA0546/2014 (Seal 1) and A/Seal/ Sweden/SVA0824/2014 (Seal 2), which were detected 4.5 months apart, possessed almost identical HA and NA genes (99% identity).The HA and NA genes clustered within the Eurasian avian lineage (Figure 2 and 3) showing 99% nucleotide similarity to the HA and NA genes of a German seal isolate from September 2014, A/harbour seal/Germany/1/2014/H10N7, using the BLAST programme of the Global Initiative on Sharing All Influenza Data (GISAID)s EpiFlu Database (http:// www.gisaid.org).These sequences were obtained from GISAID's EpiFlu database (details are given at the end of the article).The amino acid sequence at the cleavage site in the HA molecule was PELVQGR/GLF, characteristic of low-pathogenicity AIV.
IAV was isolated from lung tissue of Seal 2 using specific pathogen-free (SPF) embryonated hens' eggs (EE) as previously described [8].The allantoic fluid from the first passage in the EE showed haemagglunitating activity (HA > 256).The haemagglutinating agent could be further identified as influenza A(H10N7) using specific reference antisera for H1-H16 and N1-N9 in haemagglutination inhibition and neuraminidase inhibition test (Table) [9].The virus had an intravenous pathogenicity index [8] value of 0.00, confirming the low pathogenicity of the virus for chickens.

Discussion
Although IAV infection has been reported in a variety of species of marine mammals including seals [10][11][12], this is, to our knowledge, the first published report of AIV isolation from seals in Europe and the first time that the H10 subtype has been detected in seals anywhere.It provides evidence that the H10N7 subtype was associated with an outbreak of seal mortality in Europe.Although we detected the virus in only two affected seals, media reports support H10N7 involvement in seal mortality events in Denmark, Germany and the Netherlands, as the virus was isolated from numerous dead seals [1][2][3].
As in AIV-associated mortality events in seals in the United States (US), Seal 1 suffered from a concurrent bacterial pneumonia [10].Viral damage to physical components of the respiratory immune system is thought to allow secondary invasion of opportunistic bacteria.Limited quantity and quality of material from Seal 2 precluded investigation of bacterial infection.
Through phylogenetic analyses, we showed that this virus is genetically closely related to Eurasian AIVs from wild and domestic birds (Figure 2 and 3).IAVs are known to be circulating at high prevalence in European aquatic birds [13], supporting initial introduction of the H10 virus in seals from aquatic birds in Europe.
The seals probably contracted the virus through direct or indirect contact with wild birds or their droppings.Interspecies transmission from birds to seals From a public health perspective, extended circulation within a mammalian host not only demonstrates that this strain is capable of infecting and circulating in mammals, but it increases the opportunity for mutations to occur that may facilitate human infection.For example, the H3N8 strain from harbour seals in the US had recent mutations that are known to make influenza viruses more transmissible and cause more severe disease [12].It also has the ability to target the SAα-2,6 receptor found in the human respiratory tract, an adaptation known to increase transmission and virulence in mammalian hosts [12].In addition, some avian H10 viruses, including those isolated from farmed mink (Mustela vison) (H10N4), humans (H10N7, H10N8) and pigs (H10N5) (Figure 2) had the unique ability to cause severe disease in mammalian species without prior adaptation in poultry, supporting the hypothesis that these viruses in particular might pose a threat to human health [14][15][16][17][18][19][20].
Outbreaks of diseases among marine mammals can also involve interaction with humans and wild and domesticated animals, therefore, circulation of AIVs in mammals may have potential implications for public health.Management of dead marine mammals is challenging and especially difficult when they carry a new pathogen with unknown infectivity for humans.We lack information on the zoonotic potential of this particular strain of AIV and highlight the need for further assessment and research regarding risks for public health.
Handling and disposal of carcasses may expose people to any number of potential zoonotic pathogens.This necessitates applying the precautionary principle as well as close collaboration and sharing of responsibility and resources between agencies at the local and national level for situations in which jurisdictional boundaries are often poorly defined.

Nucleotide sequences accession number
The nucleotide sequences obtained in the present study have been made available in the database of the Global Initiative on Sharing All Influenza Data (GISAID) under accession numbers EPI545212, EPI545213 (Seal 1) and EPI547696, EPI547697 (Seal 2).For the phylogenetic analysis, relevant sequences were obtained from the influenza database of the National Center for Biotechnology Information (NCBI).We also acknowledge the authors, originating and submitting laboratories for the HA and NA sequences of A/harbour seal/ Germany/1/2014 with accession numbers EPI544351 and EPI544353 from GISAID's EpiFlu Database.

Figure 3
Figure 3Phylogenetic relationship between neuraminidase genes of N7 influenza A virus subtypes 6.0.Numbers below key nodes indicate the percentage of bootstrap values of 2,000 replicates.Isolates sequenced in this study are indicated by a red dot.Taxons leading to A(H10) subtype viruses detected in mammalian species are highlighted in pink.The nucleotide sequences obtained in the present study are available in the Global Initiative on Sharing All Influenza