A Silent Enzootic of an Orthopoxvirus in Ghana, West Africa: Evidence for Multi-Species Involvement in the Absence of Widespread Human Disease

Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera (Cricetomys, Graphiurus, Funiscirus, and Heliosciurus) had antibodies against OPXV, and three genera (Cricetomys, Graphiurus, and Xerus) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurring OPXV infection, and that persons living near forests may have low-level or indirect exposure to OPXV-infected animals, possibly resulting in sub-clinical infections

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Abstract. Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera ( Cricetomys , Graphiurus , Funiscirus , and Heliosciurus ) had antibodies against OPXV, and three genera ( Cricetomys , Graphiurus , and Xerus ) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurring OPXV infection, and that persons living near forests may have low-level or indirect exposure to OPXV-infected animals, possibly resulting in sub-clinical infections. * Address correspondence to Mary G. Reynolds, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-43 Atlanta, GA 30333. E-mail: [email protected] † These authors contributed equally to this article. 747 ORTHOPOXVIRUSES IN GHANA other practices that could have influenced virus transmission among captive animals. Two localities outside Accra were identified by the facility owner as being the areas from which the original animals from the April 2003 shipment had been collected. One site (5.98125N, 0.58489E) was near the town of Sogakofe, in the Volta region and the other site (5.78562N, 1.01410W) was near the city of Oda in the Eastern region. In March-April, 2004, animals representing several of the species contained in the 2003 consignment and others commonly found in the two source areas were collected and evaluated for the presence of MPXV and for serologic evidence of prior or current infection. The collection and processing of animals followed The CDC laboratories in Atlanta evaluated tissue specimens for evidence of OPXV and MPXV (by using a polymerase chain reaction [PCR] and virus culture) and assayed blood specimens for antibodies (by using an enzyme-linked immunosorbent assay [ELISA]). Serosurvey. Residents living in villages near the two original trapping sites were contacted by Ghanaian Ministry of Health officials and CDC investigators and asked to participate in a research study to evaluate human exposure to OPXV species. Local Ministry of Health workers explained the purpose of the study and read the consent form to prospective enrollees, translating from English when necessary. Parental consent was obtained for persons less than 18 years of age. Adolescents (age = 11-17 years) and children (age = 7-10 years) were asked to provide their assent to participate in the study. Persons who consented to participate were asked to provide basic demographic information (e.g., age, occupation) and to answer several questions pertaining to illness history, smallpox vaccination status, and exposure to wildlife. In addition, one blood specimen was collected from each study participant. Blood for serologic testing was collected in standard marbletop vacutainer™ tubes and stored refrigerated for not more than 48 hours before being centrifuged. Centrifugation and collection of serum was conducted at the Noguchi Memorial Institute for Medical Research. Serum specimens were divided and frozen; one aliquot was kept at the Ministry of Health laboratories and one was sent to CDC for analysis of OPXVreactive IgG and IgM. Persons who participated were compensated for their time and effort. The protocol for this study was reviewed and approved by institution review boards at the CDC in Atlanta (CDC protocol #4043) and at the Noguchi Memorial Institute for Medical Research in Accra. Laboratory methods. Detailed methods for preparation and analysis of tissues using real-time PCR are described elsewhere. 11 All processing of animal tissues was performed under BioSafety Level 3 conditions. Solid tissues (pooled or individual) were placed in disposable dounce homogenizers (Kendall Large Tissue Grinders; Kendall Company catalog no. 3500SA; Tyco Healthcare, Mansfield, MA). One milliliter of phosphate-buffered saline was added to each measured tissue sample before grinding to enable creation of slurries. Genomic DNA was prepared from an aliquot of each slurry (kit catalog no. 732-6340; Bio-Rad, Hercules, CA). Remaining tissue slurries were stored for future virus isolation (see below). Nucleic acid samples prepared from pooled tissues were tested by real-time PCR for OPXV DNA before testing individual tissues. The primer/probe sequences were selected from the DNA polymerase gene (E9L; GenBank accession no. L22579) with Primer Express version 1.5 (Applied Biosystems, Foster City, CA). These sequences included OPX forward primer (5′-TCA AAT ATT GAT CGT CCA ACG A-3′), OPX reverse primer (5′-TGG ATG AAT TTC TCA ATA TTA GTT GG-3′), and OPX probe (5′FAM-TAA CAT CCG TCT GGA GAT ATC CCG TTA GA-BHQ1-3′). Primers and probe were synthesized in the Biotechnology Core Facility (CDC) by using standard phosphoramidite chemistry. Each reaction (25 μL) contained 5 μL of template DNA, 0. were one cycle at 95°C for 10 minutes and 45 cycles at 95°C for 15 seconds and 60°C for 1 minute. The PCR amplification is based on fluorescent emission after annealing/elongation (60°C). Each reaction in the real-time assay was performed in triplicate, and the operator was blinded to the identity of the source DNA. Vaccinia virus Wyeth DNA (50 fg) was used as a positive control and six wells with water were used as negative controls. In all wells, the ABI TaqMan exogenous internal positive control DNA (Applied Biosystems) and primers/probe mixture was included to assay for PCR inhibition that may be caused by the sample. Any tissue pool showing a positive result in the assay was further analyzed at the level of individual tissue (i.e., samples of kidney, eyelid, tongue, brain, blood, heart, liver, gonad, lesion, oral swab, ocular swab, lymph node, feces, skin, and urine). Specimens from individual tissues that had positive results were also analyzed for MPXV-specific DNA signatures (targeting the B6R gene encoding an envelope protein 12 ), and an aliquot of the sample slurry was used for virus isolation on cultured BSC-40 cells. Reproducibly positive samples were additionally analyzed for a secondary OPXV DNA sequence (14-kD fusion protein, A27L) by using the Artus Orthopox LC PCR kit (Qiagen, Valencia, CA). 13 Tissue specimens for which positive results were not reproducible were scored as equivocal; further analysis was not attempted on these specimens. A modified OPXV ELISA was used for animal screening. 11, 14 Human serum samples were assessed for IgG and IgM reactive with OPXV antigen in accordance with published methods. 14 Antibody positivity was measured and compared 748 REYNOLDS, CARROLL AND OTHERS with reference standards. Any reading that was three standard deviations above the mean of the negative controls was inferred to be positive. Statistical analyses. Epidemiologic and demographic variables and qualitative laboratory findings were analyzed by using parametric and nonparametric statistical tests. Nonparametric statistical tests were used when analyzing subsets of data that were not normally distributed. The Mantel-Haenszel common odds ratio (OR) and Fisher's exact tests (two-tailed) were used for categorical variables, and the Student's t -test was used for comparison of means derived from continuous variables. A P value < 0.050 was used to measure significance of associations. RESULTS Investigation of export facility. Interviews with the U.S. importer and the licensed commercial Ghanaian exporter indicated that in March-April 2003 collections of multiple Ghanaian mammal species were made by local persons. These animals were subsequently shipped to the United States. The shipment included several rodent species including Gambian rats ( Cricetomys spp.), rope squirrels ( Funisciurus spp.), dormice ( Graphiurus spp.), sun squirrels ( Heliosciurus spp.), striped mice ( Lemiscomys barbarus ), and brush-tailed porcupines ( Atherurus africanus ). These collections were made gradually over a period of a few weeks. While awaiting shipment, the pouched rats were stored together in concrete enclosures and the squirrels were housed in contiguous wire mesh cages with several individuals per cage. All animals were grouped with members of their own species. Most of the squirrels, and pouched rats were collected in a forest zone northwest of Accra The animal shipments were packed into multi-tiered wooden crates with wire-lined compartments. Members of each of the smaller species ( Lemniscomys and Graphiurus ) were shipped with approximately 25 animals per compartment. The exporter reported seeing nothing unusual as far as overt disease in the animals before shipment, nor were any unusual animal dieoffs seen at his facility before shipment. The shipment was sent as freight aboard a commercial airline and entered the United States through Dallas, Texas. Animals collected during the investigation. To determine which species of interest might be capable of harboring MPXV in nature, members of the Ghanaian Division of Wildlife Services, CDC, the U.S. Department of Agriculture, and local trappers collected 204 animals over nine nights during March 24-April 2, 2004. Approximately 28% (57 animals) were collected in the Eastern region and the remaining from the Volta region Among 182 animals for which serologic analysis was performed, only 2 (one a Cricetomys sp. and the other a Funisciurus sp.) had detectable levels of antibodies against OPXV at the highest reciprocal serum dilution of 400 Using the molecular screening algorithm outlined above, we found that 6 of 97 Graphiurus , 2 of 38 Cricetomys , and 1 of 1 Xerus showed evidence of OPXV DNA signatures in harvested tissue specimens Tissues from the nine animals that showed positive results by real-time PCR specific for OPXV (generic) polymerase (E9L) DNA signatures were also assayed for a secondary OPXV DNA target (14-kD fusion protein). Three of the nine animals were also positive for this secondary OPXV ( Xerus 289, Graphiurus 389, and Graphiurus 431) ( 12, 13 A cytopathic effect caused by OPXV was not observed in virus cultures prepared from the tissues of any animals, nor was culturable virus obtained after serial passage. (In our hands, virus culture is typically 10-100-fold less sensitive than the most sensitive real-time PCR in detecting OPXV in clinical specimens.) One Graphiurus spp. (animal number 431) had a low level of antibodies against OPXV in its serum and OPXV DNA signatures in its spleen. Two Funisciurus spp. with antibodies Human serosurvey. To investigate whether persons working closely with sylvan animals and humans living near animal trapping sites might be at enhanced risk for exposure to OPXVs, we conducted a serosurvey among residents of several villages within walking distance of where the animals implicated in the U.S. outbreak had been originally collected. For purposes of this study, these villages will be referred to as villages A/B (Eastern region) and Village C (Volta region). In all, 65 residents of village C (approximately 92% of the total village population), 100 residents of villages A/B (approximately 33% and 85% of the total village populations, respectively), and 7 persons who were directly involved in the exotic animal trade consented to participate in the serosurvey Most village residents reported that they entered the forest at least once a week (98% and 95% for villages A/B and C, respectively), whereas five of six of those actively engaged in the animal trade reported only going into the forest on a monthly basis. Cultivation and firewood collection were cited most frequently as the principal reason for participants going into forested areas (n = 76 [71%] and n = 28 [26%], respectively), among those 107 participants who provided a response to the question. A higher proportion of men than women cited cultivation as the reason for entering the forest (67% versus 33%, respectively); this bias was of similar proportion in the Eastern and Volta regions. Other reasons cited for forest entry were animal trapping related activities (n = 2) and accompanying adults (n = 1). All enrolled study participants provided blood for quantification of IgG and IgM against OPXV. Serologic tests used in this study do not detect specific antibodies against MPXV but do detect antibodies broadly cross-reactive against antigens from multiple OPXVs, including MPXV and vaccinia virus (the live virus component of the smallpox vaccine). None of the study participants had positive results for IgM against OPXV, which suggested that none had been exposed to an OPXV (for which they had been previously immunologically negative) within the prior two months. 14 However, 91 (53%) study participants had detectable levels of IgG against OPXV The proportion of adults (greater than 23 years of age) with positive findings for IgG against OPXV was relatively similar Eastern 2 9 27 5 0 0 0 1 38 Lemniscomys 24 Volta 0 0 24 0 0 0 0 0 20 Tatera 9 Volta 0 0 9 0 0 0 0 0 9 Funiscirus 9 Eastern 0 3 6 0 1 0 0 1 5 Mus 8 Volta 0 0 8 0 0 0 0 0 8 Heliosciurus 7 Eastern 0 0 4 0 1 0 0 0 7 Arvicanthis 6 Volta 0 1 6 0 0 0 0 0 4 Xerus 1 Eastern 1 0 0 100 0 0 0 0 1 * PCR = polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay. † The orthopoxvirus DNA detection threshold for the orthopoxvirus generic E9L polymerase real-time PCR assay is ≥ 2 fg of purified vaccinia DNA. ‡ Result replicated in independent assay; represents finding from liver, spleen, skin, and/or pooled organ specimens. Details are shown in 750 REYNOLDS, CARROLL AND OTHERS in study participants from the Eastern and Volta regions (87% and 77%, respectively). However, among persons ≤ 23 years of age, there was a significantly greater probability of a positive finding for IgG against OPXV among those living in the Eastern region (OR = 4.05, 95% confidence interval [CI] = 1.50-11.03) ( Evaluation of absolute levels of IgG against OPX in the study population showed that in persons ≤ 23 years of age, levels 16 Although substantially lower than the mean OD-COV for persons acutely infected with MPXV and persons one year post-infection, the mean OD-COV for IgG-positive Ghanaians ≤ 23 years of age (OD-COV = 0.17) was significantly higher than that for OPXVnegative persons in the United States (OD-COV = −0.05; P < 0.001 by two-sided Student's t -test, degrees of freedom = 41). Although overall levels of IgG against OPXV among IgGpositive persons in this study were low relative to what is typically observed within one year of acute OPXV infection, six persons in this study showed highly elevated levels of IgG against OPXV (defined here as OD-COV > 2 standard deviations above the mean of the IgG-positive population in the study). All of these persons were greater than 23 years of age and described themselves as farmers. Five were from the Volta region; they reported having received smallpox vaccinations. One was from the Eastern region; this person did not report receiving a smallpox vaccination and had no evidence of a vaccine-associated scar. DISCUSSION Somewhat contrary to expectations, our results did not implicate a single species of animal as being the sylvan source of MPXV for the U.S. outbreak. We found evidence suggestive of intermittent exposure to OPXVs among various species across a broad geographic span. Species of animal showing evidence of OPXV exposure (i.e., Cricetomys spp., Graphiurus spp., Funiscuris spp., Xerus spp.) mirrored those implicated in the U.S. outbreak, 11 with the exception of the Xerus spp., which had not previously been associated with OPXV exposure. It should be emphasized that our study was not intended to constitute a comprehensive survey of the sylvan fauna of Ghana for evidence of OPXV infection. Only rodent species were evaluated. Species implicated in the U.S. outbreak were preferentially collected. Many of the species were sampled below the threshold needed to determine whether their OPXV nucleic acid and seroprevalence rates were comparable to those observed for Graphiurus spp., the most well sampled species (for which nucleic acid and antibody prevalence rates of 6.2% and 10%, respectively, were observed). If we assume that rodents, like humans, will sustain life-long detectable OPXV antibody titers after infection, the relatively 16 This figure appears in color at www.ajtmh.org

The pox in the North American backyard: Volepox virus pathogenesis in California mice (Peromyscus californicus).

Volepox virus (VPXV) was first isolated in 1985 from a hind foot scab of an otherwise healthy California vole (Microtus californicus). Subsequent surveys in San Mateo County, CA, revealed serological evidence suggesting that VPXV is endemic to this area, and a second viral isolate from a Pinyon mouse (Peromyscus truei) was collected in 1988. Since then, few studies have been conducted regarding the ecology, pathology, and pathogenicity of VPXV, and its prevalence and role as a potential zoonotic agent remain unknown. To increase our understanding of VPXV disease progression, we challenged 24 California mice (Peromyscus californicus) intranasally with 1.6 × 10(3) PFU of purified VPXV. By day five post infection (pi) we observed decreased activity level, conjunctivitis, ruffled hair, skin lesions, facial edema, and crusty noses. A mortality rate of 54% was noted by day eight pi. In addition, internal organ necrosis and hemorrhages were observed during necropsy of deceased or euthanized animals. Viral loads in tissues (brain, gonad, kidney, liver, lung, spleen, submandibular lymph node, and adrenal gland), bodily secretions (saliva, and tears), and excretions (urine, and/or feces) were evaluated and compared using real time-PCR and tissue culture. Viral loads measured as high as 2 × 10(9) PFU/mL in some organs. Our results suggest that VPXV can cause extreme morbidity and mortality within rodent populations sympatric with the known VPXV reservoirs

Viable virus content per specimen.

<p>Volepox virus loads (PFU/mL) per specimen taken during the necropsy of deceased and euthanized mice on days 6, 7 and 8 pi. <b>*</b>Swabs from the survivors were taken on day 7 pi.</p

Characteristic lesions observed on <i>P.californicus</i> infected with Volepox virus.

<p>Epidermal ulcers with crusting on paw (A), vulvae and perineum (B), tongue and nares (C). Serosal petechiae and focal gastrointestinal tract necrosis (D).</p

Immunohistochemical test results showing abundant volepox antigen.

<p>The antigen stained in red (arrow heads) within epithelial cells of a stomach specimen.</p

Hematoxylin-eosin stain of liver tissue.

<p>The arrow head indicates an example of cell necrosis (hepatocytes with homogeneously eosinophilic, or pink, cytoplasm and pyknotic or karyolytic nuclei). Hepatocytes occasionally have small basophilic intracytoplasmic inclusions observed in dark blue (arrow).</p

Electron microscopic examination of the inclusion bodies.

<p>Three types of ATIs were observed; inclusions containing virions throughout (Fig. 5A), inclusions without virions (Fig. 5B), and inclusions with virions at the periphery (Fig. 5C). The ATIs examined had varying morphologies that included both non-condensed and mature virions inside and/or around the periphery of the inclusions (Fig. 5D, E). B-type inclusions (BTIs) were also observed (Fig. 5F). The arrow head in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043881#pone-0043881-g005" target="_blank">figure 5A, and 5D</a>, shows a mature volepox virion; the arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043881#pone-0043881-g005" target="_blank">figure 5D, and 5E</a>, show immature or non-condensed virions.</p