Vero and VeroDogSLAM (VDS) cells were grown in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum. A blood sample from an infected seal from the 1988 epizootic was obtained from Albert Osterhaus, Erasmus University (Rotterdam, the Netherlands). Brain tissue from a harbor seal (designated PDV/3541UK) that was found off the coast of Scotland at the end of the 2002 epizootic and was PCR positive for PDV in the brain but not other tissues (lung, spleen, and lymph nodes) was obtained from Paul Jepson, Institute of Zoology, Zoological Society of London.

Total RNA was extracted from infected cells and tissues by using TRIzol reagent (Invitrogen). cDNA synthesis was conducted by using oligo-dT primers and the SuperScript First-Strand Synthesis kit (Invitrogen). PCR was performed by using the High Fidelity Taq kit (Invitrogen). Morbillivirus universal P gene and β-actin primers (14) and further PDV primers to the H, F, M, and P genes designed to previously published PDV sequences are given in the Table (Table). DNA sequencing was performed by using a BigDye 3.1 Terminator Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) with primers listed in the Table. Completed PCR products were sent to the Genomics Core Facility, Queens University (Belfast, UK) for chromatographic preparation.

Signaling lymphocyte activation molecule (SLAM) is a receptor for both vaccine and wild-type strains of measles virus and for canine distemper and rinderpest morbilliviruses (15,16). VDS cells have been used successfully to isolate PDV from experimentally infected ferrets (Mustela putorius furo) (17), and we recently confirmed that this molecule also is used as a receptor for PDV (M. Melia et al., unpub. data). The USA 2006 virus was isolated by inoculating homogenized liver tissue from a harbor seal onto VDS cells, which resulted in syncytia formation (Figure 1). Reverse transcription–PCR (RT-PCR) was initially conducted by using previously published morbillivirus universal P gene primers (14). The PCR product was sequenced and aligned with morbillivirus sequences for this target region and showed 100% homology with PDV (data not shown).

Selected gene sequences from the PDV/Ulster 88, PDV/NL88, PDV/DK 88, and PDV/DK2002 viruses from Europe were available for comparison with the PDV/USA2006 strain (10,18–21). In addition, we included partial P gene sequences obtained from tissues and nasal swabs of northern seas otters (Enhydra lutris) from an unusual mortality event in south-central Alaska in 2006, as well as samples from a harp seal found in the Gulf of St. Lawrence (Canada) in 1991 and from a hooded seal on the New Jersey, USA, coast in 1998 (22,23).

Because of improvements in techniques of sequence determination from those available in 1988, we initially resequenced the PDV/Ulster88 virus H, M, and F genes. We have designated this “new” sequence PDV/Ulster88n. For the same reason, and to determine whether adaptation to Vero cells changed the virus sequence, particularly in the cell receptor attachment H protein, we obtained RNA directly from a blood sample used to isolate the PDV/NL88 strain and conducted RT-PCR directly. We designated this “new” sequence for the H, F, and M genes as PDV/NL88n. The sequence obtained from the central nervous system tissue of the seal from the 2002 epizootic is designated PDV/3541UK.

Previous studies have shown that the H protein is the most variable and that comparison of the PDV/2002 and PDV88 sequences from Europe showed 8 minor amino acid changes (10,21). The sequences for the PDV/Ulster88 and PDV/NL88 viruses had been obtained from isolates in Vero (African green monkey kidney) cells, unlike the 2002 sequences, which were obtained directly from lung tissue. To determine whether errors may have occurred in the PDV/Ulster88 sequence, we initially compared the original PDV/Ulster88 and new PDV/Ulster88n H gene sequences. Changes in PDV/Ulster88 compared with the consensus sequence for all the viruses at bases 910, 911, 1134, and 1135 are not mirrored in the PDV/Ulster88n sequence, which indicates that these are likely to have resulted from sequencing errors and therefore do not reflect real differences in the more recent isolates. Similarly, to determine whether changes in the original PDV/NL88 strain are likely to have resulted from adaption to Vero cells, we compared the original PDV/NL88 H sequence with PDV/NL88n, which was amplified directly from a blood sample. Differences of PDV/NL88 from the consensus at bases 23 and 1711 are not reflected in the PDV/NL88n sequence, which indicates that these are likely to have resulted from tissue culture adaptation and/or sequencing errors.

The newly isolated PDV/USA2006 virus has 11 aa changes in the H gene (GenBank accession no. 1375698), compared with the PDV/DK2002 strain at codons 176, 200, 218, 221, 276, 327, 399, 432, 561, 561, and 576. However, the sequences at codons 200, 218, 276, 399, and 432 are in common with the PDV/NL88n strain. Similarly, silent mutations at nt 180, 564, and 1728 are the same as PDV/NL88n. With the exception of codon 564, all other differences of PDV/USA2006, compared with PDV/DK2002, reflect the consensus sequence. All of these observations suggest that the similarities of PDV/USA2006 to the 1988 virus may be due to circulation of multiple lineages. A phylogenic analysis for the H gene is shown in Figure 2, panel A.

Few amino acid changes are found in the P (GenBank accession no. 1375683) and M (Figure 3) genes of PDV/USA2006, compared with the other viruses. Complete P gene sequences were available for the DK2002, NL2002, Ulster, NL and DK 88 viruses. Partial sequences from the 1991 Canadian and 1998 USA viruses, as well as the sea otter 2006 virus, also were used for comparison (22,23). The USA/2006 P gene differs from the more recent 2002 viruses at codon 152 but is the same as all three 1988 isolates. Other minor variations between strains at the sequence level align PDV/USA2006 most closely to PDV/NL88. A phylogenic analysis based on the P gene is shown in Figure 2, panel B, and a concatenated tree based on both the H and P genes (for viruses where both sequences are available) is shown in Figure 2, panel C. The M and F gene sequence of PDV/USA2006 were compared with the sequences of PDV/Ulster88n, PDV/NL88n, PDV/DK88, and PDV/3541UK. The PDV/USA2006 M and F protein amino acid sequences (Figures 3, 4) reflect at least 1 of the other viruses at all positions. PDV/Ulster 88 M sequence differs from the other viruses at codon 84, whereas PDV/NL88n differs at codons 178, 310, 331, and 333. The first 233 nt of the PDV/3541UK M gene could not be amplified. However, alignment of the remaining sequence showed amino acids to be in common with >1 of the other viruses. In the F gene, an amino acid change occurred in the initiation codon from Met to Val in PDV/3541UK, compared with the other viruses, and silent mutations occurred at codons 20, 22, 51, 156, 198, 440, and 528.