Serologic Evidence for Novel Poxvirus in Endangered Red Colobus Monkeys, Western Uganda

Enzyme-linked immunosorbent assay, Western blot, and virus neutralization assays indicated that red colobus monkeys in Kibale National Park, western Uganda, had antibodies to a virus that was similar, but not identical, to known orthopoxviruses. The presence of a novel poxvirus in this endangered primate raises public health and conservation concerns.

chemically immobilized in the fi eld by intramuscular injection of tiletamine/zolezepam (4.6-9.6 mg/kg body weight) with 1.0-or 1.5-mL plastic darts with 5/8-inch needles shot from a variable-pressure compressed air rifl e. After initial darting, animals were given tiletamine/zolezepam or ketamine HCl intravenously as needed. Blood samples were collected into vacutainers containing sodium-EDTA, plasma was separated in the fi eld by centrifugation, and samples were stored in liquid nitrogen for transport to North America. Animals were placed in cloth bags to recover from anesthesia and were released near trees and vines that were easy to climb and within visual range of their social groups. All animals appeared healthy at the time of capture. Animal protocols were approved by the McGill University Animal Care Committee before data collection.
A vaccinia virus (VV) ELISA was used as an initial screening test to detect antipoxvirus antibodies. VV-reactive antibodies were detected with horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rhesus macaque (RM; Macaca mulatta) immunoglobulin (Ig) G (Fc specific; Nordic Laboratories, Tilsbug, the Netherlands), which readily detected antibodies of other primate species, including red colobus monkeys and humans. By using this approach, samples from 8 (26%) of 31 red colobus plasma Postadsorption ELISA tests (7) were used next to determine the specifi city of the response to 1 of 3 orthopoxviruses: VV, monkeypox virus (MPV), or cowpox virus (CPV). Plasma samples were tested directly by ELISA or preadsorbed (1:30) for 30 minutes at 37°C with VV, MPV, or CPV lysate normalized to contain 6 × 10 8 PFU/mL before addition to virus-coated ELISA plates ( Figure 1). The postadsorption ELISA test allows one to differentiate among closely related orthopoxviruses. For instance, VV-specifi c antibodies are best depleted by VV lysate assayed on a VVcoated ELISA plate (Figure 1, panel A). However, analysis of samples from 10 red colobus with detectable anti-VV antibody responses (online Appendix Figure) did not show a clear pattern following preadsorption with VV, MPV, or CPV antigens, which suggests a similar cross-reactivity to each of these 3 orthopoxviruses.
Western blot analysis was performed to characterize more fully the antipoxvirus response of the red colobus. Two micrograms of purifi ed MPV, VV, and CPV viral proteins were separated by 4%-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difl uoride membranes (Invitrogen, Carlsbad, CA, USA), and probed with plasma (1:10,000) before addition of HRP-conjugated polyclonal goat antihuman Ig G (γ-specifi c, Jackson Immunolabs, Inc., West Grove, PA, USA) and chemiluminescent detection (Pierce SuperSignal West Dura Substrate, Rockville, IL, USA) ( Figure 2). VV-immune human, MPV-immune human, MVA (modifi ed vaccinia Ankara)-immune RM and MPV-immune RM plasma samples were included as positive controls to identify banding patterns typically observed in orthopoxvirusimmune humans and nonhuman primates. Western blot analysis proved more sensitive than anti-VV ELISA (online Appendix Figure), with plasma from 30 of 31 red colobus reacting with at least 1 protein band from MPV, VV, or CPV. However, unlike the orthopoxvirus-immune human and RM controls, samples from red colobus demonstrated fewer immunoreactive bands and different immunodominant banding patterns, suggesting infection with either a distantly related orthopoxvirus or a virus from a different genus in the Poxviridae family.
Members of the Orthopoxvirus genus elicit cross-neutralizing antibodies against other members of the same genus. To determine if the red colobus were infected with an orthopoxvirus or a more distantly related poxvirus, plaque reduction neutralization assays were performed using 100 PFU of MPV, VV, or CPV. Plasma samples from all 31 red colobus were tested, and all exhibited a neutralizing titer 50 of <20 against MPV, VV, or CPV (data not shown). These fi ndings suggest that although the monkeys were infected with a poxvirus with serologic cross-reactivity to VV (online Appendix Figure), lack of a detectable neutralizing antibody response (<20) indicates that the animals may have been infected with a poxvirus that is not a member of the Orthopoxvirus genus.

Conclusions
Our results provide evidence that red colobus in Kibale National Park have been exposed to a previously uncharacterized poxvirus. Kibale red colobus may have been exposed to monkeypox or to a "monkeypox-like" virus, but we could not confi rm this with our current serologic tools. On the other hand, other poxviruses, such as Tanapox virus and Yaba monkey tumor virus, have been identifi ed in Africa, and infection by 1 of these poxviruses or a related virus cannot be ruled out. Future studies will require optimizing serologic diagnostics against these divergent poxviruses (with appropriate positive and negative controls) to determine the identity of the poxvirus/poxviruses that have infected the red colobus described here. In this light, we  note that tanapox, a zoonosis of suspected primate origin (8,9), derives its name from the Tana River, eastern Kenya, which supports an isolated population of red colobus closely related to those in Kibale (P. r. rufomitratus) (10,11). A protracted outbreak of infectious disease occurred in Kibale red colobus from 1971 to 1981, where it caused a death rate up to nearly 10% in some social groups, apparently killing only adult male monkeys (12). Although neither formal clinical data nor biologic samples were collected, descriptions of lesions of affected monkeys suggested diffuse to multifocal areas of infl ammation with gray mottling and epidermal crusts on the face (most commonly the lips), perineum, and inguinum, followed by alopecia and impaired locomotion. Monkeys sampled for the present study would almost certainly not yet have been born during this period, but these observations raise the possibility that outbreaks of disease at least outwardly consistent with poxvirus infection have occurred previously in Kibale red colobus.
Poxviruses are known for their potential to cross species barriers (1), and red colobus living in small, unprotected forest fragments outside of Kibale National Park interact aggressively and at high rates with local persons and their domestic animals (13). At the same time, persons in rural western Uganda already bear a high incidence of pathogens, including HIV (14), which renders a substantial proportion of the population immunocompromised and susceptible to opportunistic infections. Recent outbreaks of zoonotic poxviruses have not been documented in our study area, despite a local and regional healthcare system that would most likely have detected such events. However, the presence of a novel and potentially zoonotic poxvirus in red colobus should be viewed as a point of concern for the future of public health in this region and elsewhere.