A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

Abstract

Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA's Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo. These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV resulted in a single mutation in the region encoding the glycoprotein (GP). This is a region where mutations can have important consequences on outbreaks and human disease [S. Mahanty and M. Bray, Lancet Infect Dis 4:487-498, 2004, https://doi.org/10.1016/S1473-3099(04)01103-X]. We thus investigated whether this mutation impacted disease by using a cynomolgus macaque model of MARV infection. Monkeys exposed to virus containing the mutation had better clinical outcomes than monkeys exposed to virus without the mutation. We also observed that a remarkably low number of MARV particles was sufficient to cause death. Our results could have a significant impact on how future studies are designed to model MARV disease and test vaccines and therapeutics.

Keywords: Marburg virus; adaptation; attenuation; glycoprotein; monkey model; pathogenesis; signal peptide.

FIG 1

Percentage of abundance of reads containing SNP in the MARV GP gene in populations over passage in Vero E6 cell culture. MARV was serially passaged in Vero E6 cells at three different MOI (0.001, 0.01, and 0.1). Deep sequencing was used to investigate genotypic changes that occurred after cell culture passaging. Graphs display the relative abundance of reads containing cytosine (C) at the SNP locus site, after each passage. (A) MARV was passaged 10 times at an MOI of 0.001. (B) The first five passages were repeated at the same MOI (0.001). (C) The first five passages were repeated at two different higher MOI (0.01 and 0.1).

FIG 2

Changes in MARV particle/PFU ratio after cell culture passage in Vero E6 cells. Passage 2 MARV was serially passaged 10 times in Vero E6 cells at an MOI of 0.001. (A) After each passage, the number of virus particles per milliliter was determined via TEM (singlicate). (B) After each passage, the viral titer (PFU per milliliter) was determined via plaque assay (singlicate). (C) After each passage, the particle/PFU ratio was determined based on the titer and number of particles.

FIG 3

Survival proportions of NHPs exposed to MARV. Shown are survival proportions of animals exposed to either 100 or 0.01 PFU of MARV that was either low passage number (P3) or high passage number (P13). Results from individual animals are displayed.

FIG 4

Serum and tissue titers in NHPs exposed to MARV. (A) At each scheduled sedation, blood was collected from all NHPs, and serum was later processed to determine levels of infectious virus in the blood. Serum titers (PFU per milliliter) for all NHPs throughout the course of the study are shown. (B) Serum titers (PFU per milliliter) for animals exposed to the 100-PFU group of either P3 or P13 virus are shown. Means with standard deviations (SD) are shown. (C) When animals were necropsied, samples of tissue were taken and later analyzed for viral load. Titers (PFU per gram) from selected tissues for all NHPs are shown.

FIG 5

Clinical scores and temperature in MARV-exposed NHPs. (A) Animals were observed at least twice daily, and clinical scores were recorded. Clinical scores from the morning observation periods for all NHPs throughout the course of the study are shown. (B) At each scheduled sedation, rectal temperatures (°C) were taken. Rectal temperatures for all NHPs throughout the course of the study are shown.

FIG 6

Lymphocyte and neutrophil percentages in MARV-exposed NHPs. During each scheduled sedation, blood specimens were collected and analyzed. Results of hematology analysis for all NHPs throughout the course of the study are shown. (A) Percentage of lymphocytes. (B) Percentage of neutrophils.

FIG 7

Clinical chemistry parameters in MARV-exposed NHPs. During each scheduled sedation, blood specimens were collected and analyzed. Results of clinical chemistry analysis for all NHPs throughout the course of the study are shown. (A) ALT. (B) ALP. (C) GGT. (D) BUN. (E) ALB.

FIG 8

Coagulation parameters in NHPs exposed to MARV. During each scheduled sedation, blood specimens were collected and analyzed. Results of clinical coagulation analysis for all NHPs throughout the course of the study are shown. (A) PT. (B) aPTT. (C) Platelets.