The Effect of Plasmodium on the Outcome of Ebola Virus Infection in a Mouse Model

The Effect of Plasmodium on the Outcome of Ebola Virus Infection in a Mouse Model Abstract Following the Ebola virus epidemic in West Africa, several studies investigated whether there was an effect of Plasmodium coinfection on survival in Ebola virus (EBOV) disease patients. Different effects of coinfection were found in different patient cohorts. To determine whether an effect of Plasmodium coinfection on EBOV survival may exist, we modeled coinfection of Plasmodium yoelii and mouse-adapted EBOV (MA-EBOV) in CD1 mice. Subsequent infection with MA-EBOV at different time points after P. yoelii infection did not have any significant effect on survival. Ebola virus, Plasmodium, malaria, coinfection, survival Many laboratories providing diagnostics to Ebola treatment centers during the West African Ebola epidemic simultaneously performed diagnostic testing for malaria. This enabled researchers to assess whether coinfection of Ebola virus (EBOV)–infected patients with Plasmodium species parasites affected disease outcome. Interestingly, findings in the different patient cohorts ranged from a negative effect, to no effect, to a positive effect on patient survival when comparing patients infected with EBOV alone to patients coinfected with EBOV and Plasmodium species. The first study to describe an association between Plasmodium species coinfection and EBOV survival was based on a patient cohort from Monrovia, where survival of EBOV-infected patients increased from 46% in patients infected with EBOV alone to 58% in patients coinfected with EBOV and Plasmodium species [1]. This was quickly followed by a study from Guinea, where there was a negative effect of Plasmodium species coinfection when the cohort was age-stratified; the case-fatality rate increased 20% in patients between 5 and 14 years of age, but no effect of coinfection was observed in other age groups [2]. This finding was not corroborated in a small cohort of pediatric patients in Liberia and Sierra Leone, where no effect of Plasmodium species coinfection on EBOV survival was detected [3]. Another small cohort of patients from Guinea also did not find a statistically significant effect of Plasmodium species coinfection on survival [4]. A negative effect on survival in EBOV-infected patients coinfected with Plasmodium species was observed in a cohort from Sierra Leone, with a case-fatality rate of 66% in coinfected patients vs 52% in patients infected with EBOV alone [5]. The last study, based on deep sequencing of patient material, found that mortality in EBOV-infected patients increased with an increase in Plasmodium species sequence reads; however, there was no statistical correlation [6]. In a first attempt to investigate experimentally whether Plasmodium species coinfection has an effect on survival during EBOV infection, we combined 2 established mouse models for EBOV and Plasmodium, using mouse-adapted EBOV (MA-EBOV) and Plasmodium yoelii coinfection in CD1 mice. All animal experiments were approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories, National Institutes of Health, and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility, according to the institution’s guidelines for animal use, and followed the guidelines and basic principles in the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. Sample inactivation was performed according to standard operating procedures for removal of specimens from high containment approved by the Institutional Biosafety Committee. We chose the P. yoelii 17XNL strain for our experiments, as it is nonlethal in mice [7] and any mortality in coinfected mice must thus be due to the MA-EBOV infection. We first performed a time-course study, with 7 groups of 4 CD1 mice inoculated intraperitoneally with 104 parasitized erythrocytes (PE), a dose that was determined to produce the most consistent parasitemia in these mice over time in a previous dose-finding study (data not shown). The infection dynamics of P. yoelii in the blood of mice inoculated with 104 PE were followed over time. Very low levels of parasitized red blood cells (RBCs) were observed on 3 days postinoculation (dpi), which steadily increased over time, peaking in individual animals at 40%–50% parasitized RBCs between 9 and 18 dpi (Figure 1A). The increase in parasitized RBCs coincided with a decrease in the number of RBCs until 15–18 dpi (Figure 1B). Starting on 6 dpi, clear increases in lymphocytes, neutrophils, and monocytes in the blood of infected mice were observed (Figure 1C). Figure 1. View largeDownload slide Kinetics of Plasmodium yoelii infection in CD1 mice. Seven groups of 4 CD1 mice were inoculated intraperitoneally with 104 parasitized erythrocytes of Plasmodium yoelii. The percentage of parasitized red blood cells (RBCs) in inoculated mice was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (A). Every third day after inoculation, 1 group of mice was euthanized, and a blood sample collected for analysis in an IDEXX ProCyte DX hematology analyzer. The number of RBCs (B), lymphocytes, neutrophils, and monocytes (C) are plotted over time. Figure 1. View largeDownload slide Kinetics of Plasmodium yoelii infection in CD1 mice. Seven groups of 4 CD1 mice were inoculated intraperitoneally with 104 parasitized erythrocytes of Plasmodium yoelii. The percentage of parasitized red blood cells (RBCs) in inoculated mice was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (A). Every third day after inoculation, 1 group of mice was euthanized, and a blood sample collected for analysis in an IDEXX ProCyte DX hematology analyzer. The number of RBCs (B), lymphocytes, neutrophils, and monocytes (C) are plotted over time. Since the timing of coinfection with Plasmodium species in relation to EBOV infection was unknown in the described patient cohorts, we assessed whether there was an effect of P. yoelii coinfection on MA-EBOV survival in CD1 mice by inoculating 8 groups of 10 mice with 104 PE P. yoelii strain 17XNL and subsequently coinfecting them intraperitoneally with 100 median lethal dose of MA-EBOV at different times post–P. yoelii inoculation. From each group, 6 animals were used to observe survival and 4 mice were used for a scheduled necropsy 4 days after MA-EBOV infection. The first group was coinfected with MA-EBOV immediately after inoculation with P. yoelii, and an additional group was coinfected every third day after P. yoelii infection. In the group that was coinfected with MA-EBOV immediately after inoculation with P. yoelii, 1 of 6 mice survived. However, none of the mice coinfected at later time points survived the MA-EBOV infection (Figure 2A). On 28 dpi, serum was collected from the surviving mouse in the group coinfected on 0 dpi and tested positive for EBOV antibodies in a virus-like particle-based enzyme-linked immunosorbent assay up to a 1:1600 dilution (data not shown), indicating that the animal was infected with MA-EBOV. Before inoculation with MA-EBOV and 4 days after inoculation with MA-EBOV, blood smears were collected to confirm P. yoelii infection and to follow parasitemia levels over time; in animals inoculated with P. yoelii alone, blood smears were collected every third day after P. yoelii inoculation for comparison. No clear differences in the number of infected RBCs were observed between the groups; the observed differences in late time points were due to 1 or 2 animals in each group clearing the P. yoelii infection slower than the rest of the group (Figure 2B). On day 4 after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected for virological and parasitological analysis. No statistically significant differences in MA-EBOV RNA copies in blood or liver were detected between the MA-EBOV–only infected group and the coinfected groups (Figure 2C). Plasmodium yoelii RNA copy numbers in blood and liver remained high throughout most of the experiment, but started to decrease around 22 dpi (Figure 2C). Figure 2. View largeDownload slide The effect of coinfection with Plasmodium yoelii (P.y.) on survival of mouse-adapted (MA) Ebola virus (EBOV) infection in CD1 mice. Eight groups of 10 mice were inoculated intraperitoneally with 104 parasitized erythrocytes (PE) of P. yoelii and subsequently coinfected intraperitoneally with 100 median lethal dose (LD50) of MA-EBOV starting immediately after inoculation with P. yoelii in the first group and every third day after P. yoelii inoculation in the remaining groups. One group of mice was inoculated with P. yoelii alone, 1 group of mice was inoculated with MA-EBOV alone, and a control group of 3 CD1 mice inoculated with MA-EBOV alone was included at every MA-EBOV inoculation time point from 3 days postinoculation (dpi) onward as an inoculum control. Six mice in each group were observed for survival (A). Animals were weighed daily and euthanized when they met predetermined humane endpoint criteria; the time of MA-EBOV inoculation is indicated at the top of each graph. Surviving mice were euthanized 28 dpi. Every third day after P. yoelii inoculation and at 4 days after MA-EBOV in the coinfected groups, the percentage of parasitized RBCs was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (B). Four days after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected, and RNA was extracted to determine the number of P. yoelii and MA-EBOV RNA copies in reverse-transcription quantitative polymerase chain reaction (PCR) as described previously [9] (C). RNA copy numbers were calculated by running standards with known copy numbers, as determined by droplet digital PCR, in parallel in each run. The 0 dpi time point of coinfection was repeated in a large group of CD1 mice inoculated intraperitoneally with 104 PE P. yoelii and subsequently coinfected intraperitoneally with 100 LD50 of MA-EBOV. A group of 10 mice inoculated with P. yoelii alone and a group of 10 mice inoculated with MA-EBOV alone were used as controls. Survival of mice over time is plotted (D). Figure 2. View largeDownload slide The effect of coinfection with Plasmodium yoelii (P.y.) on survival of mouse-adapted (MA) Ebola virus (EBOV) infection in CD1 mice. Eight groups of 10 mice were inoculated intraperitoneally with 104 parasitized erythrocytes (PE) of P. yoelii and subsequently coinfected intraperitoneally with 100 median lethal dose (LD50) of MA-EBOV starting immediately after inoculation with P. yoelii in the first group and every third day after P. yoelii inoculation in the remaining groups. One group of mice was inoculated with P. yoelii alone, 1 group of mice was inoculated with MA-EBOV alone, and a control group of 3 CD1 mice inoculated with MA-EBOV alone was included at every MA-EBOV inoculation time point from 3 days postinoculation (dpi) onward as an inoculum control. Six mice in each group were observed for survival (A). Animals were weighed daily and euthanized when they met predetermined humane endpoint criteria; the time of MA-EBOV inoculation is indicated at the top of each graph. Surviving mice were euthanized 28 dpi. Every third day after P. yoelii inoculation and at 4 days after MA-EBOV in the coinfected groups, the percentage of parasitized RBCs was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (B). Four days after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected, and RNA was extracted to determine the number of P. yoelii and MA-EBOV RNA copies in reverse-transcription quantitative polymerase chain reaction (PCR) as described previously [9] (C). RNA copy numbers were calculated by running standards with known copy numbers, as determined by droplet digital PCR, in parallel in each run. The 0 dpi time point of coinfection was repeated in a large group of CD1 mice inoculated intraperitoneally with 104 PE P. yoelii and subsequently coinfected intraperitoneally with 100 LD50 of MA-EBOV. A group of 10 mice inoculated with P. yoelii alone and a group of 10 mice inoculated with MA-EBOV alone were used as controls. Survival of mice over time is plotted (D). Although only 1 of 6 mice in the group coinfected immediately after P. yoelii inoculation survived, this was in line with the increase in survival of 20% observed in the patient cohort from Monrovia [1]. To determine whether this increased survival in mice was indeed due to the coinfection, we repeated this time point with a group of 40 coinfected mice. Two groups of 10 mice infected with MA-EBOV alone or P. yoelii alone were used as controls. Infection with P. yoelii was confirmed in all animals via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds on 4 dpi. All of the mice infected with MA-EBOV alone or coinfected with P. yoelii and MA-EBOV reached humane endpoint criteria (Figure 2D), indicating that there was no beneficial effect of P. yoelii coinfection on MA-EBOV survival. Taken together, the data presented here do not support a role of Plasmodium species coinfection in survival of EBOV infection. However, it remains to be determined whether this is due to limitations to the experimental models used here. Lethality of EBOV in mice requires the use of a mouse-adapted variant derived from the 1995 EBOV Mayinga isolate, rather than an EBOV Makona isolate from the West African epidemic. Moreover, as opposed to the P. yoelii model used here, most patients in the West African cohorts likely were not undergoing a primary Plasmodium species infection. Since the immune response to Plasmodium species in humans differs between primary and repeat infections (reviewed in [8]), the mouse model of P. yoelli may not provide the best representation of the immune status of Plasmodium species–infected individuals in West Africa. Thus, although no positive or negative effect of Plasmodium coinfection on survival of EBOV infection was shown here, more research is necessary to definitively prove the absence of such an effect. Notes Supplement sponsorship. This work is part of a supplement sponsored by xxx. Financial support. This work was funded by the Intramural Research Program of the NIAID, NIH. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Rosenke K , Adjemian J , Munster VJ , et al. Plasmodium parasitemia associated with increased survival in Ebola virus-infected patients . Clin Infect Dis 2016 ; 63 : 1026 – 33 . Google Scholar CrossRef Search ADS PubMed 2. Kerber R , Krumkamp R , Diallo B , et al. Analysis of diagnostic findings from the European mobile laboratory in Gueckedou, Guinea, March 2014 through March 2015 . J Infect Dis 2016 ; 214 : S250 – 7 . Google Scholar CrossRef Search ADS PubMed 3. Smit MA , Michelow IC , Glavis-Bloom J , Wolfman V , Levine AC . Characteristics and outcomes of pediatric patients with Ebola virus disease admitted to treatment units in Liberia and Sierra Leone: a retrospective cohort study . Clin Infect Dis 2017 ; 64 : 243 – 9 . Google Scholar CrossRef Search ADS PubMed 4. Vernet MA , Reynard S , Fizet A , et al. Clinical, virological, and biological parameters associated with outcomes of Ebola virus infection in Macenta, Guinea . JCI Insight 2017 ; 2 : e88864 . Google Scholar CrossRef Search ADS PubMed 5. Waxman M , Aluisio AR , Rege S , Levine AC . Characteristics and survival of patients with Ebola virus infection, malaria, or both in Sierra Leone: a retrospective cohort study . Lancet Infect Dis 2017 ; 17 : 654 – 60 . Google Scholar CrossRef Search ADS PubMed 6. Carroll MW , Haldenby S , Rickett NY , et al. Deep sequencing of RNA from blood and oral swab samples reveals the presence of nucleic acid from a number of pathogens in patients with acute Ebola virus disease and is consistent with bacterial translocation across the Gut . mSphere 2017 ; 2 . doi: 10.1128/mSphereDirect.00325-17 . 7. Fu Y , Ding Y , Zhou TL , Ou QY , Xu WY . Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii . J Parasitol 2012 ; 98 : 310 – 5 . Google Scholar CrossRef Search ADS PubMed 8. Crompton PD , Moebius J , Portugal S , et al. Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease . Annu Rev Immunol 2014 ; 32 : 157 – 87 . Google Scholar CrossRef Search ADS PubMed 9. de Wit E , Munster VJ , Rosenke K , et al. Ebola laboratory response at ELWA, Monrovia, Liberia 2014–2015 . J Infect Dis 2016 ; 214 ( Suppl 3 ): S169 – 76 . Google Scholar CrossRef Search ADS PubMed Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

The Effect of Plasmodium on the Outcome of Ebola Virus Infection in a Mouse Model

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Abstract

Abstract Following the Ebola virus epidemic in West Africa, several studies investigated whether there was an effect of Plasmodium coinfection on survival in Ebola virus (EBOV) disease patients. Different effects of coinfection were found in different patient cohorts. To determine whether an effect of Plasmodium coinfection on EBOV survival may exist, we modeled coinfection of Plasmodium yoelii and mouse-adapted EBOV (MA-EBOV) in CD1 mice. Subsequent infection with MA-EBOV at different time points after P. yoelii infection did not have any significant effect on survival. Ebola virus, Plasmodium, malaria, coinfection, survival Many laboratories providing diagnostics to Ebola treatment centers during the West African Ebola epidemic simultaneously performed diagnostic testing for malaria. This enabled researchers to assess whether coinfection of Ebola virus (EBOV)–infected patients with Plasmodium species parasites affected disease outcome. Interestingly, findings in the different patient cohorts ranged from a negative effect, to no effect, to a positive effect on patient survival when comparing patients infected with EBOV alone to patients coinfected with EBOV and Plasmodium species. The first study to describe an association between Plasmodium species coinfection and EBOV survival was based on a patient cohort from Monrovia, where survival of EBOV-infected patients increased from 46% in patients infected with EBOV alone to 58% in patients coinfected with EBOV and Plasmodium species [1]. This was quickly followed by a study from Guinea, where there was a negative effect of Plasmodium species coinfection when the cohort was age-stratified; the case-fatality rate increased 20% in patients between 5 and 14 years of age, but no effect of coinfection was observed in other age groups [2]. This finding was not corroborated in a small cohort of pediatric patients in Liberia and Sierra Leone, where no effect of Plasmodium species coinfection on EBOV survival was detected [3]. Another small cohort of patients from Guinea also did not find a statistically significant effect of Plasmodium species coinfection on survival [4]. A negative effect on survival in EBOV-infected patients coinfected with Plasmodium species was observed in a cohort from Sierra Leone, with a case-fatality rate of 66% in coinfected patients vs 52% in patients infected with EBOV alone [5]. The last study, based on deep sequencing of patient material, found that mortality in EBOV-infected patients increased with an increase in Plasmodium species sequence reads; however, there was no statistical correlation [6]. In a first attempt to investigate experimentally whether Plasmodium species coinfection has an effect on survival during EBOV infection, we combined 2 established mouse models for EBOV and Plasmodium, using mouse-adapted EBOV (MA-EBOV) and Plasmodium yoelii coinfection in CD1 mice. All animal experiments were approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories, National Institutes of Health, and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility, according to the institution’s guidelines for animal use, and followed the guidelines and basic principles in the US Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. Sample inactivation was performed according to standard operating procedures for removal of specimens from high containment approved by the Institutional Biosafety Committee. We chose the P. yoelii 17XNL strain for our experiments, as it is nonlethal in mice [7] and any mortality in coinfected mice must thus be due to the MA-EBOV infection. We first performed a time-course study, with 7 groups of 4 CD1 mice inoculated intraperitoneally with 104 parasitized erythrocytes (PE), a dose that was determined to produce the most consistent parasitemia in these mice over time in a previous dose-finding study (data not shown). The infection dynamics of P. yoelii in the blood of mice inoculated with 104 PE were followed over time. Very low levels of parasitized red blood cells (RBCs) were observed on 3 days postinoculation (dpi), which steadily increased over time, peaking in individual animals at 40%–50% parasitized RBCs between 9 and 18 dpi (Figure 1A). The increase in parasitized RBCs coincided with a decrease in the number of RBCs until 15–18 dpi (Figure 1B). Starting on 6 dpi, clear increases in lymphocytes, neutrophils, and monocytes in the blood of infected mice were observed (Figure 1C). Figure 1. View largeDownload slide Kinetics of Plasmodium yoelii infection in CD1 mice. Seven groups of 4 CD1 mice were inoculated intraperitoneally with 104 parasitized erythrocytes of Plasmodium yoelii. The percentage of parasitized red blood cells (RBCs) in inoculated mice was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (A). Every third day after inoculation, 1 group of mice was euthanized, and a blood sample collected for analysis in an IDEXX ProCyte DX hematology analyzer. The number of RBCs (B), lymphocytes, neutrophils, and monocytes (C) are plotted over time. Figure 1. View largeDownload slide Kinetics of Plasmodium yoelii infection in CD1 mice. Seven groups of 4 CD1 mice were inoculated intraperitoneally with 104 parasitized erythrocytes of Plasmodium yoelii. The percentage of parasitized red blood cells (RBCs) in inoculated mice was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (A). Every third day after inoculation, 1 group of mice was euthanized, and a blood sample collected for analysis in an IDEXX ProCyte DX hematology analyzer. The number of RBCs (B), lymphocytes, neutrophils, and monocytes (C) are plotted over time. Since the timing of coinfection with Plasmodium species in relation to EBOV infection was unknown in the described patient cohorts, we assessed whether there was an effect of P. yoelii coinfection on MA-EBOV survival in CD1 mice by inoculating 8 groups of 10 mice with 104 PE P. yoelii strain 17XNL and subsequently coinfecting them intraperitoneally with 100 median lethal dose of MA-EBOV at different times post–P. yoelii inoculation. From each group, 6 animals were used to observe survival and 4 mice were used for a scheduled necropsy 4 days after MA-EBOV infection. The first group was coinfected with MA-EBOV immediately after inoculation with P. yoelii, and an additional group was coinfected every third day after P. yoelii infection. In the group that was coinfected with MA-EBOV immediately after inoculation with P. yoelii, 1 of 6 mice survived. However, none of the mice coinfected at later time points survived the MA-EBOV infection (Figure 2A). On 28 dpi, serum was collected from the surviving mouse in the group coinfected on 0 dpi and tested positive for EBOV antibodies in a virus-like particle-based enzyme-linked immunosorbent assay up to a 1:1600 dilution (data not shown), indicating that the animal was infected with MA-EBOV. Before inoculation with MA-EBOV and 4 days after inoculation with MA-EBOV, blood smears were collected to confirm P. yoelii infection and to follow parasitemia levels over time; in animals inoculated with P. yoelii alone, blood smears were collected every third day after P. yoelii inoculation for comparison. No clear differences in the number of infected RBCs were observed between the groups; the observed differences in late time points were due to 1 or 2 animals in each group clearing the P. yoelii infection slower than the rest of the group (Figure 2B). On day 4 after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected for virological and parasitological analysis. No statistically significant differences in MA-EBOV RNA copies in blood or liver were detected between the MA-EBOV–only infected group and the coinfected groups (Figure 2C). Plasmodium yoelii RNA copy numbers in blood and liver remained high throughout most of the experiment, but started to decrease around 22 dpi (Figure 2C). Figure 2. View largeDownload slide The effect of coinfection with Plasmodium yoelii (P.y.) on survival of mouse-adapted (MA) Ebola virus (EBOV) infection in CD1 mice. Eight groups of 10 mice were inoculated intraperitoneally with 104 parasitized erythrocytes (PE) of P. yoelii and subsequently coinfected intraperitoneally with 100 median lethal dose (LD50) of MA-EBOV starting immediately after inoculation with P. yoelii in the first group and every third day after P. yoelii inoculation in the remaining groups. One group of mice was inoculated with P. yoelii alone, 1 group of mice was inoculated with MA-EBOV alone, and a control group of 3 CD1 mice inoculated with MA-EBOV alone was included at every MA-EBOV inoculation time point from 3 days postinoculation (dpi) onward as an inoculum control. Six mice in each group were observed for survival (A). Animals were weighed daily and euthanized when they met predetermined humane endpoint criteria; the time of MA-EBOV inoculation is indicated at the top of each graph. Surviving mice were euthanized 28 dpi. Every third day after P. yoelii inoculation and at 4 days after MA-EBOV in the coinfected groups, the percentage of parasitized RBCs was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (B). Four days after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected, and RNA was extracted to determine the number of P. yoelii and MA-EBOV RNA copies in reverse-transcription quantitative polymerase chain reaction (PCR) as described previously [9] (C). RNA copy numbers were calculated by running standards with known copy numbers, as determined by droplet digital PCR, in parallel in each run. The 0 dpi time point of coinfection was repeated in a large group of CD1 mice inoculated intraperitoneally with 104 PE P. yoelii and subsequently coinfected intraperitoneally with 100 LD50 of MA-EBOV. A group of 10 mice inoculated with P. yoelii alone and a group of 10 mice inoculated with MA-EBOV alone were used as controls. Survival of mice over time is plotted (D). Figure 2. View largeDownload slide The effect of coinfection with Plasmodium yoelii (P.y.) on survival of mouse-adapted (MA) Ebola virus (EBOV) infection in CD1 mice. Eight groups of 10 mice were inoculated intraperitoneally with 104 parasitized erythrocytes (PE) of P. yoelii and subsequently coinfected intraperitoneally with 100 median lethal dose (LD50) of MA-EBOV starting immediately after inoculation with P. yoelii in the first group and every third day after P. yoelii inoculation in the remaining groups. One group of mice was inoculated with P. yoelii alone, 1 group of mice was inoculated with MA-EBOV alone, and a control group of 3 CD1 mice inoculated with MA-EBOV alone was included at every MA-EBOV inoculation time point from 3 days postinoculation (dpi) onward as an inoculum control. Six mice in each group were observed for survival (A). Animals were weighed daily and euthanized when they met predetermined humane endpoint criteria; the time of MA-EBOV inoculation is indicated at the top of each graph. Surviving mice were euthanized 28 dpi. Every third day after P. yoelii inoculation and at 4 days after MA-EBOV in the coinfected groups, the percentage of parasitized RBCs was determined via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds (B). Four days after inoculation with MA-EBOV, 4 mice from each group were euthanized; blood and liver were collected, and RNA was extracted to determine the number of P. yoelii and MA-EBOV RNA copies in reverse-transcription quantitative polymerase chain reaction (PCR) as described previously [9] (C). RNA copy numbers were calculated by running standards with known copy numbers, as determined by droplet digital PCR, in parallel in each run. The 0 dpi time point of coinfection was repeated in a large group of CD1 mice inoculated intraperitoneally with 104 PE P. yoelii and subsequently coinfected intraperitoneally with 100 LD50 of MA-EBOV. A group of 10 mice inoculated with P. yoelii alone and a group of 10 mice inoculated with MA-EBOV alone were used as controls. Survival of mice over time is plotted (D). Although only 1 of 6 mice in the group coinfected immediately after P. yoelii inoculation survived, this was in line with the increase in survival of 20% observed in the patient cohort from Monrovia [1]. To determine whether this increased survival in mice was indeed due to the coinfection, we repeated this time point with a group of 40 coinfected mice. Two groups of 10 mice infected with MA-EBOV alone or P. yoelii alone were used as controls. Infection with P. yoelii was confirmed in all animals via microscopy of Giemsa-stained blood smears prepared from tail vein bleeds on 4 dpi. All of the mice infected with MA-EBOV alone or coinfected with P. yoelii and MA-EBOV reached humane endpoint criteria (Figure 2D), indicating that there was no beneficial effect of P. yoelii coinfection on MA-EBOV survival. Taken together, the data presented here do not support a role of Plasmodium species coinfection in survival of EBOV infection. However, it remains to be determined whether this is due to limitations to the experimental models used here. Lethality of EBOV in mice requires the use of a mouse-adapted variant derived from the 1995 EBOV Mayinga isolate, rather than an EBOV Makona isolate from the West African epidemic. Moreover, as opposed to the P. yoelii model used here, most patients in the West African cohorts likely were not undergoing a primary Plasmodium species infection. Since the immune response to Plasmodium species in humans differs between primary and repeat infections (reviewed in [8]), the mouse model of P. yoelli may not provide the best representation of the immune status of Plasmodium species–infected individuals in West Africa. Thus, although no positive or negative effect of Plasmodium coinfection on survival of EBOV infection was shown here, more research is necessary to definitively prove the absence of such an effect. Notes Supplement sponsorship. This work is part of a supplement sponsored by xxx. Financial support. This work was funded by the Intramural Research Program of the NIAID, NIH. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Rosenke K , Adjemian J , Munster VJ , et al. Plasmodium parasitemia associated with increased survival in Ebola virus-infected patients . Clin Infect Dis 2016 ; 63 : 1026 – 33 . Google Scholar CrossRef Search ADS PubMed 2. Kerber R , Krumkamp R , Diallo B , et al. Analysis of diagnostic findings from the European mobile laboratory in Gueckedou, Guinea, March 2014 through March 2015 . J Infect Dis 2016 ; 214 : S250 – 7 . Google Scholar CrossRef Search ADS PubMed 3. Smit MA , Michelow IC , Glavis-Bloom J , Wolfman V , Levine AC . Characteristics and outcomes of pediatric patients with Ebola virus disease admitted to treatment units in Liberia and Sierra Leone: a retrospective cohort study . Clin Infect Dis 2017 ; 64 : 243 – 9 . Google Scholar CrossRef Search ADS PubMed 4. Vernet MA , Reynard S , Fizet A , et al. Clinical, virological, and biological parameters associated with outcomes of Ebola virus infection in Macenta, Guinea . JCI Insight 2017 ; 2 : e88864 . Google Scholar CrossRef Search ADS PubMed 5. Waxman M , Aluisio AR , Rege S , Levine AC . Characteristics and survival of patients with Ebola virus infection, malaria, or both in Sierra Leone: a retrospective cohort study . Lancet Infect Dis 2017 ; 17 : 654 – 60 . Google Scholar CrossRef Search ADS PubMed 6. Carroll MW , Haldenby S , Rickett NY , et al. Deep sequencing of RNA from blood and oral swab samples reveals the presence of nucleic acid from a number of pathogens in patients with acute Ebola virus disease and is consistent with bacterial translocation across the Gut . mSphere 2017 ; 2 . doi: 10.1128/mSphereDirect.00325-17 . 7. Fu Y , Ding Y , Zhou TL , Ou QY , Xu WY . Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii . J Parasitol 2012 ; 98 : 310 – 5 . Google Scholar CrossRef Search ADS PubMed 8. Crompton PD , Moebius J , Portugal S , et al. Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease . Annu Rev Immunol 2014 ; 32 : 157 – 87 . Google Scholar CrossRef Search ADS PubMed 9. de Wit E , Munster VJ , Rosenke K , et al. Ebola laboratory response at ELWA, Monrovia, Liberia 2014–2015 . J Infect Dis 2016 ; 214 ( Suppl 3 ): S169 – 76 . Google Scholar CrossRef Search ADS PubMed Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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The Journal of Infectious DiseasesOxford University Press

Published: Jun 7, 2018

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