Intra-host Ebola viral adaption during human infection

Abstract

The onsite next generation sequencing (NGS) of Ebola virus (EBOV) genomes during the 2013-2016 Ebola epidemic in Western Africa provides an opportunity to trace the origin, transmission, and evolution of this virus. Herein, we have diagnosed a cohort of EBOV patients in Sierra Leone in 2015, during the late phase of the outbreak. The surviving EBOV patients had a recovery process characterized by decreasing viremia, fever, and biochemical parameters. EBOV genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection, including the previously described short stretches of 13 serial T>C mutations. Remarkably, within individual patients, samples collected during the early phase of infection possessed Ts at these nucleotide sites, whereas they were replaced by Cs in samples collected in the later phase, suggesting that these short stretches of T>C mutations could emerge independently. In addition, up to a total of 35 nucleotide sites spanning the EBOV genome were mutated coincidently. Our study showed the dynamic intra-host adaptation of EBOV during patient recovery and gave more insight into the complex EBOV-host interactions.

Keywords: Adaptation; Blood biochemistry; Clinical manifestations; Ebola virus; Genome sequencing; Intra-host Nucleotide Variation.

Figure 1

The diagnosis and initial clinical features of EBOV patients at hospitalization. A. Correlation of the Ct values from RT-PCR testing in patient blood samples for EBOV GP and NP. B. Average days from symptom onset to initial sampling, as categorized by survival and fatal cases. C. Comparison of temperatures during initial sampling between survival and fatal cases. D. Ct values between survival and fatal cases.

Figure 2

The longitudinal clinic variation of the EBOV patients during the disease process. The EBOV shedding and body temperature. The EBOV shedding was represented by the Ct values (blue) tested by RT-PCR targeting EBOV GP in blood. The closed boxes indicate the Ct ≤ 38 (Ebola RNA Positive); while the open boxes denote the Ct>38 or no Ct available (Ebola RNA Negative).When there are two longitudinally-collected blood samples with Ebola RNA Negative, the patient will be discharged. The corresponding temperatures (purple) during the blood sampling.

Figure 3

Blood biochemistry of Ebola patients. Blood parameters were tested at the SLE-CHN Bio-safety Lab in Sierra Leone. The range of values in healthy individuals is denoted as a pink area.

Figure 4

The coincident transition of the 35 coincident nucleotide variations of EBOV during the recovery process. A. Only nucleotide positions with >100 reads were estimated. The red and black lines below the genome structure were used to highlight the specific genomic regions of EBOV with iSNVs. B–D. The similar substitution trends of the EBOV iSNV in longitudinally collected blood samples of patients 18791, 18797 and 18889. The substitution ratio of each point = the reads of the dominant iSNV at the later time/the reads of the dominant iSNV at the early time of the disease. The 50% substitution (ratio = 1) was shown in purple dashed line. The list of the iSNV sites is present in Table2. The exceptional sites which do not have the substitution trends are shown in Supplementary Figure S1. A. Only nucleotide positions with >100 reads were estimated. The red and black lines below the genome structure were used to highlight the specific genomic regions of EBOV with iSNVs. B–D. The similar substitution trends of the EBOV iSNV in longitudinally collected blood samples of patients 18791, 18797 and 18889. The substitution ratio of each point = the reads of the dominant iSNV at the later time/the reads of the dominant iSNV at the early time of the disease. The 50% substitution (ratio = 1) was shown in purple dashed line. The list of the iSNV sites is present in Table 2. The exceptional sites which do not have the substitution trends are shown in Supplementary Figure S1.

Figure 5

Phylogenetic analysis of the newly characterized and public full-length EBOV genome sequences. A. Sequences characterized in the present study are marked in red. B. All of the strains with serial T>C mutations are marked in blue. The numbers of serial T>C mutations are given. C. Detailed phylogenetic relationships of the strains with 13 serial T>C mutations. In this panel, sequences in the same color are obtained from the same patient at different time points. Sequences marked with black stars are reported in this paper.

Figure 6

The schematic diagram for the dynamic intra-host substitution and inter-host transmission of EBOV. Base on the dynamic adaptation of Ebola virus in the patients we described herein, we further proposed the schematic model for the relationship of the human to human transmission and the dynamic adaptation of the Ebola viruses. Panel A shows the disease process of one survived patient A from the preclinical period to symptom presentation, and then to recovery. During this process, the dominant viruses in the patient are the T strain which possess the former nucleic acids in the 35 sites (Table 2), e.g. T in the 13 T>C stretch (5512–5631). There also will be emergence of the C strains during the recovery process, which possess the latter nucleic acids in the 35 sites (Table 2), e.g. C in the 13 T>C stretch (5512–5631) due to unknown reasons as we indicated in our patients. T strain virus dominates in the acute detoxification period of patients and certain patients died during this period. Thus in the general human to human transmission of Ebola virus (patient A to patient C), the transmitted viruses are T strain virus, which takes the dominates in the 1078 EBOV genomes of the West Africa outbreak publicly available from GenBank. However, we cannot exclude the possibility of the sporadic transmission of C strains in humans with close contact (A to B). Patient B, who was infected by the C strain of Ebola virus, may have a mixed quasi-species of the virus during the diseases process. This possibility requires further exploration.