This new strain of CDV is highly conserved over the period of surveillance, geographic region and host species. It appears to be widespread in the Southeast US, but samples evaluated were samples of convenience (those submitted to the diagnostic lab for CDV testing). Additional states are likely involved but more samples are required from both healthy and symptomatic animals for an epidemiological survey.

Unlike a previous report that suggests strains stemming from the America-2 lineage are the dominant “American” strain in circulation [15], at least in eastern Tennessee, this new strain was predominant in 2011–2013. We consider this to be the third “contemporary” genotype that has been detected in North America.

The first contemporary genotype described was America 2 (represented by red clade, Fig. 3). The term genotype has been used in the grouping of CDVs, but a formal analysis has yet to be published. Genotypes have been defined by the phylogenetic properties of the H-gene amino acid sequence. Strains in the same clade showing more than 95 % amino acid homology are considered to belong to the same genotype [14]. This is an arbitrary distinction, and depending on which sequences are used for comparison the 95 % difference does not always hold true when considering currently recognized genotypes. This is evident when comparing one of the European strains (GenBank Accession DQ494319) with a European Wildlife strain (GenBank Accession (DQ228166) versus the same comparison when using a different European strain (GenBank Accession Z47761) (Table 1).

Table 2 Primer sets for genome amplification Full size table

We performed phylogenetic evaluation of current US strains in comparison to our strains and discovered circulation of another clade that has already been described but not well-defined in the literature (yellow clade, Fig. 3). A virus belonging to this clade was initially detected by Pardo et al. in 2005 [16], sample 19876 (GenBank accession AY964110), detected in Missouri, USA in 2004 but was not defined as a new genotype at that time. Strains belonging to the clade represented by sample 19876 were also detected by additional researchers and suggested to be part of the European Wildlife lineage [5] or a subgroup thereof [17] (CDV outbreak in fishers in California (GenBank accession numbers JN836734-7), based on partial H gene sequencing. Strains grouping with the 19876 clade were later also detected in Mexico (Edomex strains) and the researchers defined the clade as a new genotype/lineage [4]. Based on our phylogenetic analysis, we concur the clade represented by sample 19876 should be recognized as a new genotype based on H gene divergence (Table 1) and viruses from this genotype are currently circulating in Tennessee and Texas in the US.). Therefore, in staying with the current genotype naming scheme, this clade represents America 3 (yellow clade, Fig. 3).

Based on clade groupings and hemagglutinin amino acid divergence, we suggest this new strain circulating in the Southeast also represents a new genotype and should be called America 4 (blue clade, Fig. 3). This new America 4 genotype outgroups from other clades with similar distances as each previously described genotype does from each other. SNPs between the clades outnumber SNPs within each clade, yet CDV 13–1941 and 13–2262 have fewer differences between them than all but clonally derived isolates (only 56 nucleotide differences across the two genomes). This is unexpected as these two samples came from different species of animals, at different times and in different location with no obvious route of common exposure or transmission. It is also highly conserved, supporting the hypothesis that this novel lineage is most likely contained in a stable host reservoir with breakthrough transmission to other species.

In addition to the sequence divergence from other clades, an alternate start site increasing the size of the signal peptide of the F protein has been previously reported in the Asia-2 lineage [6]; however, this new strain adds 9 additional amino acids even beyond the Asia-2 variant. It has been suggested that variations in the signal peptide region affect F protein expression and a longer signal peptide region might result in reduced F protein production and persistent infection due to evasion of the immune system [18, 19]. This observation, in addition to the highly conserved genome, fits with our hypothesis that this strain is representative of a stable reservoir. Figure 1 reveals that the hypervariable M-F region is practically identical, if not completely identical in most of the isolates typed as the new strain. This means that the two genomes we sequenced are unlikely to be coincidentally conserved and the other isolates are, in all probability, equally conserved at the genomic level.

A virus capable of infecting and transmitting in wildlife yet not causing severe disease in a large number of infected animals would be under less selective pressure and show reduced genomic divergence between isolates, as we observed in our data. Furthermore, if this strain is significantly divergent from vaccine strains (which is also shown in our data/Figures), it may be capable of vaccine escape which is supported by our data from vaccinated dogs and our serum neutralization testing. The exact mechanism of vaccine protection in dogs with respect to CDV is unknown. Therefore we do not know what mutations, or combination of mutations, give rise to vaccine escape and host adaptation for this strain. However, if it is well-adapted in wildlife, it follows that infection in domestic dogs would be inefficient and/or infrequent. Why exactly this strain is only recently causing disease in dogs if it has been in wildlife long enough to become a reservoir is unknown; it may have been around prior to our discoveries yet undetected due to lack of genetic evaluation of strains. However, the lack of diversity in a hypervariable region from multiple dogs suggests this is a newly introduced strain.

We believe the stable host reservoir is likely the endemic wildlife population. The virus is highly labile outside of the host, so cross-species transmission occasionally occurs, thus the virus could be maintained in wildlife populations between outbreaks [20]. We have detected this strain in raccoons and foxes, as well as three dogs with a history of recent direct raccoon exposure. Raccoons, which are among the most common wildlife species found in cities and towns, are considered a secondary reservoir of CDV and spillover of infection from domestic dogs with spillback from raccoons is well documented [1].

While there have recently been multiple reports of new genotypes/lineages, questions remain as to whether the genetic differences represent significant differences in antigenicity. It has been suggested that changes in the glycan shield may result in vaccine escape mechanisms. However, comparative neutralization studies have shown that fully glycosylated and de-glycosylated wild-type viruses are neutralized with equal efficiency [21]. It was also demonstrated that only 5 of the N-glycosylation sites (N149, N391, N422, N456, and N587) are actually used by the virus and this glycosylation pattern has been highly conserved in wild-type strains. This argues against a glycan shield escape mechanism for CDV, and the presence of N309 detected in this new strain and in other recent wild-type strains, as well as N584 in some Asian strains, is meaningless [21].

CDV is currently recognized as a single serotype [22]. However, preliminary evaluation of neutralization titers comparing this strain to one of the America 1 type vaccine strains showed significant differences (Fig. 5). Neutralizing antibodies are often used as a substitute marker for protection [23], with a neutralization titer of ≥ 32 generally considered protective [24]. Evaluation of the titers from this study, in light of this cut-off value, suggests all 5 dogs would not have been considered protected pre-booster, and one dog would not have been considered protected post-vaccination when evaluating neutralization titers with this new strain. All the dogs would have been considered to have a protective titer both pre- and post-vaccination when evaluating the antibody response with the Onderstepoort vaccine strain. These results should be considered from a diagnostic standpoint with regard to which virus strain or strains should be used for routine serum neutralization testing for dogs.

The extent to which antigenic variation is leading to vaccine failure is unknown. This new strain has been associated with disease in 3 properly vaccinated adult dogs. All three dogs were euthanized due to severe clinical disease. Two of the dogs were necropsied and histologic findings were consistent with CDV infection. Unfortunately, sera from these dogs were not available for antibody testing. While it is possible these dogs were vaccine non-responders, it would not be expected based on the percentage of these animals seen in the general population (approximately 1/5000 dogs, http://www.wsava.org/sites/default/files/WSAVA_OwnerGuidelines_September2010.pdf), and the fact that these dogs were all different breeds and unrelated.

Cell-mediated immunity must also be considered with regard to vaccine protection. This was not evaluated in this study, but presence of a neutralizing titer correlates with the presence of a T-cell response, though they are not well correlated quantitatively [23]. A protective cellular immunity can be present in the absence of a detectable antibody-mediated response. A strong cellular immunity causes viral elimination but a delayed or absent lymphocyte-mediated response results in viral persistence in the CNS [25]. Challenge studies with this new strain would best determine whether dogs are protected from the new strain via cross-protection or if the new strain is genetically distinct enough to evade the vaccine but that was beyond the scope of this project.

Critical CDV-specific herd immunity resulting from low vaccination rates may contribute to outbreaks [22]. Vaccine histories were unknown for some of these animals, but the majority of the cases were in dogs with incomplete vaccination or no vaccination. This suggests herd immunity is low due to reduced vaccination rates. This has likely contributed to the emergence of this new strain. Whether there is also reduced protection in vaccinated dogs as a result of extended vaccination intervals is unknown. The issue of duration of protective immunity maintained by booster vaccination every 3 years for CDV may have to be reevaluated in light of the emergence of new variants of CDV [5]. The veterinary profession should weigh the risks of side-effects due to over-vaccination against the risk of a decreased herd immunity with the re-emergence of epidemics as a consequence [26]. Maintenance of high vaccination rates using efficacious vaccines that induce a solid, resilient immunity must still be given the highest priority in control of distemper [22]. Our data suggests that ongoing surveillance should be conducted to detect and analyze new and emerging strains of CDV that may facilitate vaccine evasion.