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Postmortem Stability of Ebola Virus

Postmortem Stability of Ebola Virus Swabs were placed in 1 mL of culture medium and tissue Joseph Prescott, Trenton Bushmaker, samples were placed in 500 μL of RNAlater (QIAGEN, Robert Fischer, Kerri Miazgowicz, Valencia, CA, USA), or an empty vial for titration, before Seth Judson, Vincent J. Munster freezing at −80°C. Carcasses were placed in vented plastic The ongoing Ebola virus outbreak in West Africa has high- containers in an environmental chamber at 27°C and 80% lighted questions regarding stability of the virus and detec- relative humidity throughout the study to mimic conditions tion of RNA from corpses. We used Ebola virus–infected in West Africa (5). At the indicated time points (<9 days macaques to model humans who died of Ebola virus dis- for 2 animals and 10 weeks for 3 animals), swab and tis- ease. Viable virus was isolated <7 days posteuthanasia; sue samples were obtained and used for EBOV titration on viral RNA was detectable for 10 weeks. Vero E6 cells to quantify virus or for quantitative reverse transcription PCR (qRT-PCR) (40 cycles) to measure viral he ongoing outbreak of Ebola virus (EBOV) infection RNA, as reported (6,7). Tin West Africa highlights several questions, including Viral RNA was detectible in all swab samples and fundamental questions surrounding human-to-human trans- tissue biopsy specimens at multiple time points (Figure mission and stability of the virus. More than 20,000 cases 1). For swab samples (Figure 1, panel A), the highest of EBOV disease (EVD) have been reported, and >8,000 amount of viral RNA was in oral, nasal, and blood sam- deaths have been documented (1). Human-to-human trans- ples; oral and blood swab specimens consistently showed mission is the principal feature in EBOV outbreaks; virus positive results for all animals until week 4 for oral is transmitted from symptomatic persons or contaminated specimens and week 3 for blood, when 1 animal was corpses or by contact with objects acting as fomites (2). negative for each specimen type. Furthermore, oral swab Contact with corpses during mourning and funeral practic- specimens had the highest amount of viral RNA after the es, which can include bathing the body and rinsing family first 2 weeks of sampling, although after the 4-week sam - members with the water, or during the removal and trans- pling time point, some samples from individual animals portation of bodies by burial teams has resulted in numer- were negative. ous infections (3). In all samples, RNA was detectable sporadically for Assessing the stability of corpse-associated virus and the entire 10-week period, except for blood, which had determining the most efficient sampling methods for diag - positive results for <9 weeks. Tissue samples were more nostics will clarify the safest practices for handling bodies consistently positive within the first few weeks after eu - and the best methods for determining whether a person has thanasia (Figure 1, panel B). All samples from the liver died of EVD and presents a risk for transmission. To fa- and lung were positive for the first 3 weeks, and spleen cilitate diagnostic efforts, we studied nonhuman primates samples were positive for the first 4 weeks, at which who died of EVD to examine stability of the virus within time lung and spleen samples were no longer tested be- tissues and on body surfaces to determine the potential for cause of decay and scarcity of tissue. Muscle sample transmission, and the presence of viral RNA associated results were sporadic: a sample from 1 animal was nega- with corpses. tive at the 1-day time point and at several times through- out sampling. The Study Viable EBOV was variably isolated from swab from We studied 5 cynomolgus macaques previously included all sampling sites. Among blood samples, those from the in EBOV pathogenesis studies and euthanized because of body cavity had the highest virus titer (2 × 10 50% tis- signs of EVD and viremia. Two animals were infected with sue culture infectious doses/mL) and longest-lasting iso- EBOV-Mayinga and 3 with a current outbreak isolate (Ma- latable virus (7 days posteuthanasia) (Figure 2, panel A). kona-WPGC07) (4). Consistent with the qRT-PCR results, for swab samples, Immediately after euthanasia, multiple samples were oral and nasal sample titers were highest, followed by those collected: oral, nasal, ocular, urogenital, rectal, skin, and for blood samples, and relatively high titers were observed blood (pooled in the body cavity) swab samples and tissue <4 days posteuthanasia (Figure 2, panel B). Similar to the biopsy specimens from the liver, spleen, lung, and muscle. qRT-PCR experiments, virus titers were higher in tissue samples than in swab samples but were not as sustained; Author affiliation: National Institutes of Health, Hamilton, all tissue samples were positive at day 3 posteuthanasia but Montana, USA negative by day 4. DOI: http://dx.doi.org/10.3201/eid2105.150041 856 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 Postmortem Stability of Ebola Virus Figure 1. Presence and stability of Ebola virus RNA in deceased cynomolgus macaques. Swab (A) and tissue (B) specimen samples were obtained at the indicated time points, and viral RNA was isolated and used in a 1-step quantitative reverse transcription PCR with a primer/probe set specific for the nucleoprotein gene and standards consisting of known nucleoprotein gene copy numbers. Line plots show means of positive samples from 5 animals up to the 7 day time point and from 3 animals thereafter. Error bars indicate SD, and - indicates time points at which ≥1 animal had undetectable levels of viral RNA. Absence of a hyphen indicates that all animals had detectible levels of viral RNA. Conclusions at least until these times. Because virus titers decreased rela- The efficiency of detecting EBOV from corpse samples has tively sharply, despite sensitivity issues, it is unlikely that not been systematically studied; this information is needed viable virus persists for times longer than we measured. for interpreting results for diagnostic samples for epidemi- Humans who die of EVD typically have high levels of ologic efforts during outbreaks. We showed that viral RNA viremia, suggesting that most fresh corpses contain high is readily detectable from oral and blood swab specimens levels of infectious virus, similar to the macaques in this for <3 weeks postmortem from a monkey carcass that was study (9). Furthermore, family members exposed to EVD viremic at the time of death, in environmental conditions patients during late stages of disease or who had contact similar to those during current outbreak (5). with deceased patients have a high risk for infection (2). The stability of the target RNA used for RT-PCR is more The presence of viable EBOV and viral RNA in body flu - robust than that of viable virus because degradation of any ids of EVD patients has been studied, and oral swabbing part of the genome (or proteins and lipids) would compro- has been shown to be effective for diagnosis of EVD by mise the ability of the virus to replicate. Thus, the ability to RT-PCR compared with testing of serum samples from the isolate replicating virus in cell culture from postmortem ma- same persons (10,11). However, detection limits for diag- terials was much less sensitive than detection of viral RNA nostic swab samples are unknown for early phases of EVD, by qRT-PCR. The sensitivity for quantitating infectious vi- and blood sampling is probably more sensitive and reliable rus is probably lowered because of limitations in isolation for antemortem diagnostics and should be used whenever efficiency on cell culture and necessary dilutions of tissues possible, which has also been shown with closely related for homogenization for titration. Nonetheless, we detected Marburg virus (12). viable virus <7 days posteuthanasia in swab specimens and Although these studies included data from outbreak 3 days in tissues, and showed that infectious virus is present situations, they are limited in their sampling numbers, Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 857 DISPATCHES Figure 2. Efficiency of Ebola virus isolation from deceased cynomolgus macaques. Swab (A) and tissue (B) specimen samples were obtained at the indicated time points, and virus isolation was attempted on Vero E6 cells. Cells were inoculated in triplicate with serial dilutions of inoculum from swab specimens placed in 1 mL of medium or tissues homogenized in 1 mL of medium. The 50% tissue culture infectious dose (TCID ) was calculated by using the Spearman-Karber method (8). Line plots show means of positive samples from 5 animals to the day 9 time point. Error bars indicate SD. swabbing surfaces, and time course, and it is unknown of epidemiologic data collected for human corpses by de- how predictive they are for samples collected postmortem. termining whether a person had EVD at the time of death It is essential to stress that swab samples should be ob- and whether contact tracing should be initiated. Further- tained by vigorous sampling to acquire sufficient biologic more, viable virus can persist for >7 days on surfaces of material for testing, and development of a quality-control bodies, confirming that transmission from deceased per - PCR target (housekeeping gene target) would be beneficial sons is possible for an extended period after death. These for sample integrity assessment, which is a limitation of data are also applicable for interpreting samples collected this study. from remains of wildlife infected with EBOV, especial- In summary, we present postmortem serial sampling ly nonhuman primates, and to assess risks for handling data for EBOV-infected animals in a controlled environ- these carcasses. ment. Our results show that the EBOV RT-PCR RNA tar- get is highly stable, swabbing upper respiratory mucosa is Acknowledgments efficient for obtaining samples for diagnostics, and tissue We thank Darryl Falzarano and Andrea Marzi for use of animal biopsies are no more effective than simple swabbing for vi- carcasses upon completion of their studies and Anita Mora for rus detection. These results will directly aid interpretation providing assistance with graphics. 858 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 Postmortem Stability of Ebola Virus 6. Marzi A, Ebihara H, Callison J, Groseth A, Williams KJ, This study was supported by the Division of Intramural Geisbert TW, et al. Vesicular stomatitis virus–based Ebola vaccines Research, National Institute of Allergy and Infectious Diseases, with improved cross-protective efficacy. J Infect Dis. 2011;204 National Institutes of Health. (Suppl 3):S1066–74. http://dx.doi.org/10.1093/infdis/jir348 7. Ebihara H, Rockx B, Marzi A, Feldmann F, Haddock E, Brining D, Dr. Prescott is a research fellow in the Virus Ecology Unit at Rocky et al. Host response dynamics following lethal infection of rhesus Mountain Laboratories, Hamilton, Montana. He is currently involved macaques with Zaire ebolavirus. J Infect Dis. 2011;204 in the Ebola virus outbreak at the combined Centers for Disease Con- (Suppl 3):S991–9. http://dx.doi.org/10.1093/infdis/jir336 8. Finney DJ. Statistical method in biological assay. New York: trol and Prevention/National Institutes of Health diagnostic labora- Macmillian Publishing Co., Inc.; 1978. p. 394–8. tory, Monrovia, Liberia. His research interests include the immune 9. Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, response, transmission, and modeling of viral hemorrhagic fevers. Vincent M, et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription–PCR in an outbreak setting and References assessment of patient viral load as a predictor of outcome. J Virol. 2004;78:4330–41. http://dx.doi.org/10.1128/ 1. Centers for Disease Control and Prevention. Ebola hemorrhagic fever [cited 2015 Jan 3]. http://www.cdc.gov.ezproxy.nihlibrary.nih. JVI.78.8.4330-4341.2004 gov/vhf/ebola/ 10. Bausch DG, Towner JS, Dowell SF, Kaducu F, Lukwiya M, Sanchez A, et al. Assessment of the risk of Ebola virus transmission 2. Dowell SF, Mukunu R, Ksiazek TG, Khan AS, Rollin PE, Peters CJ. Transmission of Ebola hemorrhagic fever: a study of risk from bodily fluids and fomites. J Infect Dis. 2007;196 (Suppl 2):S142–7. http://dx.doi.org/10.1086/520545 factors in family members, Kikwit, Democratic Republic of the Congo, 1995. J Infect Dis. 1999;179(Suppl 1):S87–91. 11. Formenty P, Leroy EM, Epelboin A, Libama F, Lenzi M, Sudeck H, http://dx.doi.org/10.1086/514284 et al. Detection of Ebola virus in oral fluid specimens during out - breaks of Ebola virus hemorrhagic fever in the Republic of Congo. 3. Khan AS, Tshioko FK, Heymann DL, Le Guenno B, Nabeth P, Kerstiëns B, et al. The reemergence of Ebola hemorrhagic Clin Infect Dis. 2006;42:1521–6. http://dx.doi.org/10.1086/503836 12. Grolla A, Jones SM, Fernando L, Strong JE, Ströher U, Möller P, fever, Democratic Republic of the Congo, 1995. J Infect Dis. 1999;179(Suppl 1):S76–86. http://dx.doi.org/10.1086/514306 et al. The use of a mobile laboratory unit in support of patient 4. Hoenen T, Groseth A, Feldmann F, Marzi A, Ebihara H, management and epidemiological surveillance during the 2005 Marburg outbreak in Angola. PLoS Negl Trop Dis. 2011;5:e1183. Kobinger G, et al. Complete genome sequences of three Ebola virus isolates from the 2014 outbreak in West Africa. Genome http://dx.doi.org/10.1371/journal.pntd.0001183 Announc. 2014;2:e01331–14. http://dx.doi.org/10.1128/ genomeA.01331-14 Address for correspondence: Vincent J. Munster, Rocky Mountain 5. Ng S, Cowling B. Association between temperature, humidity and Laboratories, 903 S 4th St, Hamilton, MT 59840, USA; email: ebolavirus disease outbreaks in Africa, 1976 to 2014. Euro Surveill. munstervj@niaid.nih.gov 2014;19:pii: 20892. June 2014: Respiratory Infections Including: • Adverse Pregnancy Outcomes and Coxiella burnetii Antibodies in Pregnant Women, Denmark • Short-Term Malaria Reduction by Single-Dose Azithromycin during Mass Drug Administration for Trachoma, Tanzania • Human Polyomavirus 9 Infection in Kidney Transplant Patients • Genetic Evidence of Importation of Drug-Resistant Plasmodium falciparum to Guatemala from the Democratic Republic of the Congo • Characteristics of Patients with Mild to Moderate Primary Pulmonary Coccidioidomycosis • Rapid Spread and Diversification of Respiratory Syncytial Virus Genotype ON1, Kenya http://wwwnc.cdc.gov/eid/articles/issue/20/6/table-of-contents Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 859 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Infectious Diseases Pubmed Central

Postmortem Stability of Ebola Virus

Emerging Infectious Diseases , Volume 21 (5) – May 1, 2015

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Pubmed Central
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1080-6040
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1080-6059
DOI
10.3201/eid2105.150041
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Abstract

Swabs were placed in 1 mL of culture medium and tissue Joseph Prescott, Trenton Bushmaker, samples were placed in 500 μL of RNAlater (QIAGEN, Robert Fischer, Kerri Miazgowicz, Valencia, CA, USA), or an empty vial for titration, before Seth Judson, Vincent J. Munster freezing at −80°C. Carcasses were placed in vented plastic The ongoing Ebola virus outbreak in West Africa has high- containers in an environmental chamber at 27°C and 80% lighted questions regarding stability of the virus and detec- relative humidity throughout the study to mimic conditions tion of RNA from corpses. We used Ebola virus–infected in West Africa (5). At the indicated time points (<9 days macaques to model humans who died of Ebola virus dis- for 2 animals and 10 weeks for 3 animals), swab and tis- ease. Viable virus was isolated <7 days posteuthanasia; sue samples were obtained and used for EBOV titration on viral RNA was detectable for 10 weeks. Vero E6 cells to quantify virus or for quantitative reverse transcription PCR (qRT-PCR) (40 cycles) to measure viral he ongoing outbreak of Ebola virus (EBOV) infection RNA, as reported (6,7). Tin West Africa highlights several questions, including Viral RNA was detectible in all swab samples and fundamental questions surrounding human-to-human trans- tissue biopsy specimens at multiple time points (Figure mission and stability of the virus. More than 20,000 cases 1). For swab samples (Figure 1, panel A), the highest of EBOV disease (EVD) have been reported, and >8,000 amount of viral RNA was in oral, nasal, and blood sam- deaths have been documented (1). Human-to-human trans- ples; oral and blood swab specimens consistently showed mission is the principal feature in EBOV outbreaks; virus positive results for all animals until week 4 for oral is transmitted from symptomatic persons or contaminated specimens and week 3 for blood, when 1 animal was corpses or by contact with objects acting as fomites (2). negative for each specimen type. Furthermore, oral swab Contact with corpses during mourning and funeral practic- specimens had the highest amount of viral RNA after the es, which can include bathing the body and rinsing family first 2 weeks of sampling, although after the 4-week sam - members with the water, or during the removal and trans- pling time point, some samples from individual animals portation of bodies by burial teams has resulted in numer- were negative. ous infections (3). In all samples, RNA was detectable sporadically for Assessing the stability of corpse-associated virus and the entire 10-week period, except for blood, which had determining the most efficient sampling methods for diag - positive results for <9 weeks. Tissue samples were more nostics will clarify the safest practices for handling bodies consistently positive within the first few weeks after eu - and the best methods for determining whether a person has thanasia (Figure 1, panel B). All samples from the liver died of EVD and presents a risk for transmission. To fa- and lung were positive for the first 3 weeks, and spleen cilitate diagnostic efforts, we studied nonhuman primates samples were positive for the first 4 weeks, at which who died of EVD to examine stability of the virus within time lung and spleen samples were no longer tested be- tissues and on body surfaces to determine the potential for cause of decay and scarcity of tissue. Muscle sample transmission, and the presence of viral RNA associated results were sporadic: a sample from 1 animal was nega- with corpses. tive at the 1-day time point and at several times through- out sampling. The Study Viable EBOV was variably isolated from swab from We studied 5 cynomolgus macaques previously included all sampling sites. Among blood samples, those from the in EBOV pathogenesis studies and euthanized because of body cavity had the highest virus titer (2 × 10 50% tis- signs of EVD and viremia. Two animals were infected with sue culture infectious doses/mL) and longest-lasting iso- EBOV-Mayinga and 3 with a current outbreak isolate (Ma- latable virus (7 days posteuthanasia) (Figure 2, panel A). kona-WPGC07) (4). Consistent with the qRT-PCR results, for swab samples, Immediately after euthanasia, multiple samples were oral and nasal sample titers were highest, followed by those collected: oral, nasal, ocular, urogenital, rectal, skin, and for blood samples, and relatively high titers were observed blood (pooled in the body cavity) swab samples and tissue <4 days posteuthanasia (Figure 2, panel B). Similar to the biopsy specimens from the liver, spleen, lung, and muscle. qRT-PCR experiments, virus titers were higher in tissue samples than in swab samples but were not as sustained; Author affiliation: National Institutes of Health, Hamilton, all tissue samples were positive at day 3 posteuthanasia but Montana, USA negative by day 4. DOI: http://dx.doi.org/10.3201/eid2105.150041 856 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 Postmortem Stability of Ebola Virus Figure 1. Presence and stability of Ebola virus RNA in deceased cynomolgus macaques. Swab (A) and tissue (B) specimen samples were obtained at the indicated time points, and viral RNA was isolated and used in a 1-step quantitative reverse transcription PCR with a primer/probe set specific for the nucleoprotein gene and standards consisting of known nucleoprotein gene copy numbers. Line plots show means of positive samples from 5 animals up to the 7 day time point and from 3 animals thereafter. Error bars indicate SD, and - indicates time points at which ≥1 animal had undetectable levels of viral RNA. Absence of a hyphen indicates that all animals had detectible levels of viral RNA. Conclusions at least until these times. Because virus titers decreased rela- The efficiency of detecting EBOV from corpse samples has tively sharply, despite sensitivity issues, it is unlikely that not been systematically studied; this information is needed viable virus persists for times longer than we measured. for interpreting results for diagnostic samples for epidemi- Humans who die of EVD typically have high levels of ologic efforts during outbreaks. We showed that viral RNA viremia, suggesting that most fresh corpses contain high is readily detectable from oral and blood swab specimens levels of infectious virus, similar to the macaques in this for <3 weeks postmortem from a monkey carcass that was study (9). Furthermore, family members exposed to EVD viremic at the time of death, in environmental conditions patients during late stages of disease or who had contact similar to those during current outbreak (5). with deceased patients have a high risk for infection (2). The stability of the target RNA used for RT-PCR is more The presence of viable EBOV and viral RNA in body flu - robust than that of viable virus because degradation of any ids of EVD patients has been studied, and oral swabbing part of the genome (or proteins and lipids) would compro- has been shown to be effective for diagnosis of EVD by mise the ability of the virus to replicate. Thus, the ability to RT-PCR compared with testing of serum samples from the isolate replicating virus in cell culture from postmortem ma- same persons (10,11). However, detection limits for diag- terials was much less sensitive than detection of viral RNA nostic swab samples are unknown for early phases of EVD, by qRT-PCR. The sensitivity for quantitating infectious vi- and blood sampling is probably more sensitive and reliable rus is probably lowered because of limitations in isolation for antemortem diagnostics and should be used whenever efficiency on cell culture and necessary dilutions of tissues possible, which has also been shown with closely related for homogenization for titration. Nonetheless, we detected Marburg virus (12). viable virus <7 days posteuthanasia in swab specimens and Although these studies included data from outbreak 3 days in tissues, and showed that infectious virus is present situations, they are limited in their sampling numbers, Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 857 DISPATCHES Figure 2. Efficiency of Ebola virus isolation from deceased cynomolgus macaques. Swab (A) and tissue (B) specimen samples were obtained at the indicated time points, and virus isolation was attempted on Vero E6 cells. Cells were inoculated in triplicate with serial dilutions of inoculum from swab specimens placed in 1 mL of medium or tissues homogenized in 1 mL of medium. The 50% tissue culture infectious dose (TCID ) was calculated by using the Spearman-Karber method (8). Line plots show means of positive samples from 5 animals to the day 9 time point. Error bars indicate SD. swabbing surfaces, and time course, and it is unknown of epidemiologic data collected for human corpses by de- how predictive they are for samples collected postmortem. termining whether a person had EVD at the time of death It is essential to stress that swab samples should be ob- and whether contact tracing should be initiated. Further- tained by vigorous sampling to acquire sufficient biologic more, viable virus can persist for >7 days on surfaces of material for testing, and development of a quality-control bodies, confirming that transmission from deceased per - PCR target (housekeeping gene target) would be beneficial sons is possible for an extended period after death. These for sample integrity assessment, which is a limitation of data are also applicable for interpreting samples collected this study. from remains of wildlife infected with EBOV, especial- In summary, we present postmortem serial sampling ly nonhuman primates, and to assess risks for handling data for EBOV-infected animals in a controlled environ- these carcasses. ment. Our results show that the EBOV RT-PCR RNA tar- get is highly stable, swabbing upper respiratory mucosa is Acknowledgments efficient for obtaining samples for diagnostics, and tissue We thank Darryl Falzarano and Andrea Marzi for use of animal biopsies are no more effective than simple swabbing for vi- carcasses upon completion of their studies and Anita Mora for rus detection. These results will directly aid interpretation providing assistance with graphics. 858 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 Postmortem Stability of Ebola Virus 6. Marzi A, Ebihara H, Callison J, Groseth A, Williams KJ, This study was supported by the Division of Intramural Geisbert TW, et al. Vesicular stomatitis virus–based Ebola vaccines Research, National Institute of Allergy and Infectious Diseases, with improved cross-protective efficacy. J Infect Dis. 2011;204 National Institutes of Health. (Suppl 3):S1066–74. http://dx.doi.org/10.1093/infdis/jir348 7. Ebihara H, Rockx B, Marzi A, Feldmann F, Haddock E, Brining D, Dr. Prescott is a research fellow in the Virus Ecology Unit at Rocky et al. Host response dynamics following lethal infection of rhesus Mountain Laboratories, Hamilton, Montana. He is currently involved macaques with Zaire ebolavirus. J Infect Dis. 2011;204 in the Ebola virus outbreak at the combined Centers for Disease Con- (Suppl 3):S991–9. http://dx.doi.org/10.1093/infdis/jir336 8. Finney DJ. Statistical method in biological assay. New York: trol and Prevention/National Institutes of Health diagnostic labora- Macmillian Publishing Co., Inc.; 1978. p. 394–8. tory, Monrovia, Liberia. His research interests include the immune 9. Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, response, transmission, and modeling of viral hemorrhagic fevers. Vincent M, et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription–PCR in an outbreak setting and References assessment of patient viral load as a predictor of outcome. J Virol. 2004;78:4330–41. http://dx.doi.org/10.1128/ 1. Centers for Disease Control and Prevention. Ebola hemorrhagic fever [cited 2015 Jan 3]. http://www.cdc.gov.ezproxy.nihlibrary.nih. JVI.78.8.4330-4341.2004 gov/vhf/ebola/ 10. Bausch DG, Towner JS, Dowell SF, Kaducu F, Lukwiya M, Sanchez A, et al. Assessment of the risk of Ebola virus transmission 2. Dowell SF, Mukunu R, Ksiazek TG, Khan AS, Rollin PE, Peters CJ. Transmission of Ebola hemorrhagic fever: a study of risk from bodily fluids and fomites. J Infect Dis. 2007;196 (Suppl 2):S142–7. http://dx.doi.org/10.1086/520545 factors in family members, Kikwit, Democratic Republic of the Congo, 1995. J Infect Dis. 1999;179(Suppl 1):S87–91. 11. Formenty P, Leroy EM, Epelboin A, Libama F, Lenzi M, Sudeck H, http://dx.doi.org/10.1086/514284 et al. Detection of Ebola virus in oral fluid specimens during out - breaks of Ebola virus hemorrhagic fever in the Republic of Congo. 3. Khan AS, Tshioko FK, Heymann DL, Le Guenno B, Nabeth P, Kerstiëns B, et al. The reemergence of Ebola hemorrhagic Clin Infect Dis. 2006;42:1521–6. http://dx.doi.org/10.1086/503836 12. Grolla A, Jones SM, Fernando L, Strong JE, Ströher U, Möller P, fever, Democratic Republic of the Congo, 1995. J Infect Dis. 1999;179(Suppl 1):S76–86. http://dx.doi.org/10.1086/514306 et al. The use of a mobile laboratory unit in support of patient 4. Hoenen T, Groseth A, Feldmann F, Marzi A, Ebihara H, management and epidemiological surveillance during the 2005 Marburg outbreak in Angola. PLoS Negl Trop Dis. 2011;5:e1183. Kobinger G, et al. Complete genome sequences of three Ebola virus isolates from the 2014 outbreak in West Africa. Genome http://dx.doi.org/10.1371/journal.pntd.0001183 Announc. 2014;2:e01331–14. http://dx.doi.org/10.1128/ genomeA.01331-14 Address for correspondence: Vincent J. Munster, Rocky Mountain 5. Ng S, Cowling B. Association between temperature, humidity and Laboratories, 903 S 4th St, Hamilton, MT 59840, USA; email: ebolavirus disease outbreaks in Africa, 1976 to 2014. Euro Surveill. munstervj@niaid.nih.gov 2014;19:pii: 20892. June 2014: Respiratory Infections Including: • Adverse Pregnancy Outcomes and Coxiella burnetii Antibodies in Pregnant Women, Denmark • Short-Term Malaria Reduction by Single-Dose Azithromycin during Mass Drug Administration for Trachoma, Tanzania • Human Polyomavirus 9 Infection in Kidney Transplant Patients • Genetic Evidence of Importation of Drug-Resistant Plasmodium falciparum to Guatemala from the Democratic Republic of the Congo • Characteristics of Patients with Mild to Moderate Primary Pulmonary Coccidioidomycosis • Rapid Spread and Diversification of Respiratory Syncytial Virus Genotype ON1, Kenya http://wwwnc.cdc.gov/eid/articles/issue/20/6/table-of-contents Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 5, May 2015 859

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Emerging Infectious DiseasesPubmed Central

Published: May 1, 2015

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