This report shows that different CDV sublineages stemming from at least two genetically distant CDV lineages recently circulated through the local raccoon population. Our conclusion is based on numerous observations: differences in the lesions observed in animal tissues, possible dissimilarities of virulence between the viruses, variation in one viral phenotype in tissue culture (formation of large syncytia by the 2001 viruses), and from the results of nucleotide sequence and phylogenetic analyses. CDV is not maintained in hosts that recover from distemper, and persistent CDV infections do not occur. However, CDV infects a wide range of genera, and though each individual population may be small, the number of alternative host species may be substantial . Forest preserves around the zoo contain many species susceptible to CDV, and it appears by inference there are separate reservoirs of different CDV lineages within the area of this study.
Since past studies indicated that wild-type CDVs differed according to geographical distribution [6, 23], we initially surmised that the local CDV occasionally formed clades of highly virulent CDV variants, resulting in periodic high mortality distemper outbreaks. We also speculated that over time, highly virulent viruses would undergo extinction, and ensuing epizootics would arise from less virulent CDV variants that could affect most of the hosts without killing them. Thus, there would be an apparent oscillation (periodicity) of the mortality rates. The situation is not as straightforward, however. As shown in figures 4,5,6, at least two different CDV lineages circulated in the raccoons from 1998 – 2001. Our findings thus suggest that the outcomes of distemper might also be influenced by properties unique to different CDV lineages and their genetic variants ("strains").
The viruses from year 2001 formed syncytia in vivo and in vitro. Previously, an inverse relationship between the proficiency of syncytium formation and the level of CDV virulence was reported: the more attenuated a strain is, the higher its fusogenicity, and fusogenicity was attributed to the viral H-protein [31–34]. Therefore, the findings of this study may appear antidogmatic because increased mortality was associated with the 2001 viruses, which formed large syncytia in vivo and in vitro. However, past notions concerning the inverse relationship between fusogenicity and virulence may be imprecise. Indeed, virulent wild-type CDVs that formed syncytia in Vero cells were recently reported; the same study demonstrated that genetic changes within the H-gene were not required for CDV growth in Vero cells , as was found in this and our previous study . Also, newer studies indicate that syncytium formation by CDV requires the concerted activities of both the H- and F- proteins [36–38], and that CDV virulence is the combined affect of various proteins including the F- and H- proteins . Thus, whereas animal studies were not performed with the virus isolates of this study to directly test whether they differ in virulence, the formation of large syncytia does not rule out the possibility that the 2001 viruses are highly virulent. Noteworthy, the 2001 viruses were detected in the hippocampus and alveoli of the raccoons. Both sites were considered unusual targets of a CDV variant that was lethal to Serengeti lions, whereas CDV in dogs was said to most frequently target the brain stem and bronchi [40, 41]. It is possible that tissue localization, especially with regard to the hippocampus, correlates with virus strain. In our experience, CDV in raccoons does not preferentially target the brain stem but rather infects all portions of the brain, with the possible exception of the hippocampus. We will be able to address the question whether specific CDV strains localize in the hippocampus of raccoons as we accumulate additional data from future outbreak, and after we conduct animal tests with the viruses we isolated. In contrast, CDV targets epithelial cells, and the presence of CDV in the alveoli of raccoons with distemper is common.
H-gene phylogenetic analyses (figure 6) suggest that a viral lineage that includes CDV A75/17 (isolated in 1975)  and the 2000 and 2001 viruses had infected various species including large felids [Fig. 6 and reference 6] for at least 28 years on both coasts and a midwestern state (and thus presumably throughout the continental USA). The seemingly widespread distribution suggests that viruses stemming from this lineage may be the dominant "American" CDV currently in circulation in the continental USA. The F -, H-, and P-gene sequence analyses (figures 4,5,6) indicate that the 1998 viruses stem from a different CDV lineage that includes American CDV strains Lederle and Snyder Hill. A recent phylogenetic analysis of the P-gene by an independent laboratory that utilized some of our P-gene data generated similar results . Because they were isolated before CDV Lederle and Snyder Hill were acquired from the ATCC for this study and have distinguishable F- and H-gene sequences , it is certain that the 1998 CDV isolates are not due to laboratory contamination. Yet, phylogenetic analyses indicate that the CDV Lederle and Snyder Hill sequences are distant to, and in the case of the H-gene, ancestral to, those of the 2000 and 2001 viruses, which are as genetically distant from the 1998 viruses as they are from Snyder Hill. The source of the 1998 viruses is thus intriguing. Prior to 1997, some area raccoons were trapped, vaccinated against CDV, then released in an attempt to curtail CDV epidemics within the local raccoon population. CDV Lederle has been used as a vaccine strain in the past . The vaccine used for the raccoons, (Galaxy-D, from Schering-Plough, Kenilworth, NJ), though, was made with CDV Onderstepoort, which is easily distinguished from the 1998 viruses by F-, H-, and P-gene analyses. However, we still could not rule out the possibility that the 1998 viruses are vaccine escape viruses from a dog vaccinated with CDV Lederle. Dogs and raccoons often frequent the same feeding sites (such as refuse disposal zones) in urban areas. The possibility of reversion to virulence of attenuated CDV exists , and a vaccine escape virus was proposed as a cause of distemper in a dog in Belfast, Northern Ireland . We could not find a current manufacturer of anti-CDV vaccine in the USA that uses CDV Lederle. However, such vaccines were in distribution overseas around 1998 , and the Chicago area undergoes constant population flux, including translocation of inhabitants (and their pets) from outside of the continental USA. Related to this, the live attenuated CDV vaccine (Galaxy-D) used by the zoo up to 1997 caused vaccine-mediated distemper in different species at the zoo that had been vaccinated. For this reason, use of that particular vaccine was discontinued; instead, Purevax™, a recombinant CDV-canary pox virus vaccine (Merial, Duluth, GA) is used; the CDV insert in the canary pox virus genome is incomplete and cannot be infectious. CDV-Lederle was isolated in 1951 from a dog with encephalitis (information provided by ATCC). An alternative interpretation of our findings is that the CDV lineage that gave rise to CDV Lederle has stabilized in the local animals and is still actively circulating; more studies are needed to resolve this matter.
EMCV has been isolated or detected in raccoons before [44, 45]. However, pathogenesis was uncertain, and it was thought that raccoons are a dead-end host for this virus . It is known that mortality during an active case of distemper is increased in the presence of polymicrobial disease . For example, a lethal outcome occurs in dogs co-infected with CDV, Bordetella bronchiseptica, and Toxoplasma gondii. It is possible that the increased mortality in 2001 was due to secondary infections with EMCV; however, no lesions attributable to EMCV were observed in pathology exams of the animals of this study, and EMCV was not isolated from all of the 2001 specimens. The significance of isolating EMCV from the brain tissue of animal 01-2641 is thus uncertain.
Our findings are especially useful for the molecular epidemiology of CDV in local wildlife, as they provide a molecular basis for CDV surveillance in area wildlife. Whereas it is considered difficult to obtain field isolates of CDV, we succeeded and can now obtain complete viral genomic sequence data (it would be difficult to do so relying solely on the limited amount of archived CDV-infected tissues from the animals of this work). Taken together, we can now monitor viral genetic drift during a long-term study of CDV in local raccoons, and will be able to conduct animal studies with the newly isolated viruses. We can also clone relevant CDV virulence genes, and express and study the biochemical properties of their specific products in vitro. The baseline genetic values established here will be helpful toward the development of a contemporary field-based model (since the animals are free-ranging) for studies on the emergence, evolution, maintenance, and transmission of morbilliviruses, and the efficacy of vaccines against changing viruses.