Survival of Potential Ebola Virus Surrogates (Bacteriophages Φ6 and MS2) on PAPR Hood Material

One of the main lines of defense protecting healthcare workers from serious pathogens, like Ebola, during patient care is personal protective equipment (PPE). Personal protective equipment is wearable protection items worn by HCW to prevent the spread of disease, both from patient to HCW to patient and from patient to HCW. As PPE is barrier protection; it can become contaminated on its surface during patient care. This poses a risk when it is time to doff PPE at the end of use. Doffing is the detailed process of removing the PPE after patient care. Even with comprehensive training, self-contamination continues to occur. As self-contamination with PPE during the doffing process has been documented through several studies, the question of survivability of infectious diseases on PAPR hoods needs to be investigated. The aim of this study is to determine the recovery and survival of bacteriophages phi6 and MS2 on shroud Powered Air Purifying Respirator (PAPR) material at 45% relative humidity. Each bacteriophage was dried on shroud PAPR hood material coupons at 45% RH over several time points. Survivability was measured by utilizing the double agar layer (DAL) plaque assay method. The projected log reduction calculated from the regression lines for phi6 is estimated to have a ~1 log10 reduction at 24 hours at 45% RH, MS2 is estimated to have a ~1 log10 reduction by 96 hours. MS2 is estimated to only have a .25 log10 reduction at 24 hours at 45% RH. Both bacteriophage MS2 and phi6 survive on shroud PAPR hood material for over 24 hours at 45% relative humidity levels. The results provide evidence that both types of virus survive on PAPR hood material after the longest patient encounter, which is projected to be 8 hours. If a HCW’s PPE were to be contaminated during their patient contact time, the virus is still active once the HCW doffs their PPE and the potential for self-contamination could occur

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

Monkeypox Disease Transmission in an Experimental Setting: Prairie Dog Animal Model

Monkeypox virus (MPXV) is considered the most significant human public health threat in the genus Orthopoxvirus since the eradication of variola virus (the causative agent of smallpox). MPXV is a zoonotic agent endemic to forested areas of Central and Western Africa. In 2003, MPXV caused an outbreak in the United States due to the importation of infected African rodents, and subsequent sequential infection of North American prairie dogs (Cynomys ludovicianus) and humans. In previous studies, the prairie dog MPXV model has successfully shown to be very useful for understanding MPXV since the model emulates key characteristics of human monkeypox disease. In humans, percutaneous exposure to animals has been documented but the primary method of human-to-human MPXV transmission is postulated to be by respiratory route. Only a few animal model studies of MPXV transmission have been reported. Herein, we show that MPXV infected prairie dogs are able to transmit the virus to naive animals through multiple transmission routes. All secondarily exposed animals were infected with MPXV during the course of the study. Notably, animals secondarily exposed appeared to manifest more severe disease; however, the disease course was very similar to those of experimentally challenged animals including inappetence leading to weight loss, development of lesions, production of orthopoxvirus antibodies and shedding of similar levels or in some instances higher levels of MPXV from the oral cavity. Disease was transmitted via exposure to contaminated bedding, co-housing, or respiratory secretions/nasal mucous (we could not definitively say that transmission occurred via respiratory route exclusively). Future use of the model will allow us to evaluate infection control measures, vaccines and antiviral strategies to decrease disease transmission

Transmissibility of the monkeypox virus clades via respiratory transmission: investigation using the prairie dog-monkeypox virus challenge system.

Monkeypox virus (MPXV) is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV) related to MPXV) and cessation of routine smallpox vaccination (with the live OPXV vaccinia), there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively). Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model

0001152384.INDD

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

H&E stained lung tissue collected from a prairie dog that died due to MPXV infection (PD8021).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055488#pone-0055488-g008" target="_blank">Figure 8a</a> shows a low magnification photomicrograph of the lung (100× original magnification, scale bar = 100 micrometer). The pleural surface (arrows) is covered with an inflammatory exudate admixed with fibrin. Underneath there is a dense inflammatory infiltrate filling the alveoli and expanding alveolar walls. In the center of the image there is a foreign body type giant cell (FB). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055488#pone-0055488-g008" target="_blank">Figure 8b</a> shows a high magnification photomicrograph of the foreign body giant cell (FB) in the infiltrate in the lung (400× original magnification, scale bar = 20 micrometer).</p

Schematic showing primary challenged and secondarily infected prairie dogs’ disease progression.

<p>Transmissability of MPXV was studied within the prairie dog MPXV model. For each study, eight cages with holes on one side were utilized to individually cross-house eight animals. Schematic shows disease and molecular findings for primary challenged animal (PD8121) and cross-housed secondarily infected animal (PD8021). Arrow indicates possible infection * 2 serum samples before day 34 not available <b>¤</b> Only used lesion presentation for estimation of infection.</p