Genetic Changes at the Glycoprotein Editing Site Associated With Serial Passage of Sudan Virus

Abstract

Sudan virus (SUDV), like the closely related Ebola virus (EBOV), is a filovirus that causes severe hemorrhagic disease. They both contain an RNA editing site in the glycoprotein gene that controls expression of soluble and full-length protein. We tested the consequences of cell culture passage on the genome sequence at the SUDV editing site locus and determined whether this affected virulence. Passage resulted in expansion of the SUDV editing site, similar to that observed with EBOV. We compared viruses possessing either the wild-type or expanded editing site, using a nonhuman primate model of disease. Despite differences in virus serum titer at one time point, there were no significant differences in time to death or any other measured parameter. These data imply that changes at this locus were not important for SUDV lethality.

Keywords: RNA editing; Sudan virus; cell culture adaptation; editing site; glycoprotein.

Figure 1.

Editing site genotype of Sudan virus (SUDV) after serial passage in Vero E6 cells. SUDV was serially passaged in Vero E6 cells at a low multiplicity of infection (MOI) of 0.001 plaque-forming units (PFU)/cell. Ultra-deep sequencing was used to investigate the relative abundance of uridine (U) at the editing site after each passage. A, SUDV was passaged 18 times. B, The first 5 passages were repeated at the same MOI. C, The first 5 passages were repeated at different MOIs (0.01, 0.1, and 1.0 PFU/cell).

Figure 2.

Editing site genotype and phenotype of Sudan virus (SUDV) after infection in Macaca fascicularis. Eight M. fascicularis were challenged intramuscularly with 100 plaque-forming units of one of 2 SUDV stocks. Four animals received virus that had only been passaged in Vero E6 cell culture 3 times and had a 7-uridine (U) genotype (P3 virus). The other 4 received virus that had been passaged in Vero E6 cell culture 13 times and had an 8-U genotype (P13 virus). A, Nucleic acid was isolated from serum specimens, and ultra-deep sequencing was used to determine the sequence of the editing site in the viral RNA. B, Survival proportions of animals challenged with P3 vs P13 virus. No statistical difference was observed using the log-rank Mantel–Cox test. Abbreviation: NHPs, nonhuman primates.

Figure 3.

Serum titers and rectal temperatures in Macaca fascicularis experimentally infected with P3 or P13 Sudan virus (SUDV). Eight M. fascicularis were challenged intramuscularly with 100 plaque-forming units (PFU) of one of 2 SUDV stocks; 4 animals received virus that had a 7-uridine genotype (P3 virus), and 4 animals received virus that had an 8-uridine genotype (P13 virus). A, Blood samples were collected on days 0, 3, 5, 7, and 10 and at the time of euthanasia for viral load determination. No significant differences were found on days 3 or 5 or at time of death (P > .05, by 2-way analysis of variance with the Bonferroni post hoc test); only day 7 serum titers exhibited a significant difference (P < .01). B, At each scheduled time of blood sample collection in the morning, rectal temperature was recorded. All animals initially exhibited increases in temperature, followed by a decline. Abbreviation: NHP, nonhuman primate.

Figure 4.

Coagulation times and hematology in Macaca fascicularis experimentally infected with P3 or P13 Sudan virus (SUDV). Eight M. fascicularis were challenged intramuscularly with 100 plaque-forming units of one of 2 SUDV stocks; 4 animals received virus that had a 7-uridine genotype (P3 virus), and 4 animals received virus that had an 8-uridine genotype (P13 virus). Blood specimens were collected on days 0, 3, 5, 7, and 10 for analysis of coagulation times and hematologic testing. A, Activated partial thromboplastin time (aPTT). No data are available for animals 960 and 962 on day 7 after infection because samples did not produce readings within range for the test. Coagulation data on day 5 after infection were not recorded for animal 962. B, Percentage of neutrophils and lymphocytes. C, Number of platelets. D, Albumin levels. Abbreviation: NHP, nonhuman primate.

Figure 5.

Findings of blood chemistry analysis of Macaca fascicularis experimentally infected with P3 or P13 Sudan virus (SUDV). Eight M. fascicularis were challenged intramuscularly with 100 plaque-forming units of one of 2 SUDV stocks; 4 animals received virus that had a 7-uridine genotype (P3 virus), and 4 animals received virus that had an 8-uridine genotype (P13 virus). Blood was collected on days 0, 3, 5, 7, and 10 for blood chemistry analysis. A, Alanine aminotransferase (ALT) levels. B, Alkaline phosphatase (ALP) levels. C, γ glutamyl transferase (GGT) levels. D, Blood urea nitrogen (BUN) levels. Abbreviation: NHP, nonhuman primate.