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

Alfson, Kendra J.; Avena, Laura E.; Beadles, Michael W.; Menzie, Heather; Carrión, Ricardo; Griffiths, Anthony John

doi:10.1093/infdis/jiv216

Cited by 14 publications

(31 citation statements)

References 29 publications

(57 reference statements)

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“…Contaminating DNA was digested with the RNase-free DNase set (Qiagen). RNA samples were prepared for sequencing as previously described (66). Briefly, DNA, rRNA, and mRNA were removed from harvested nucleic acids by using a Turbo DNA-free kit (Life Technologies), a Ribo-Zero magnetic gold kit (Epicenter Biotechnologies), and RNA purification beads (Illumina), respectively.…”

Section: Construction Of Infectious Proviruses and Generation Of Virumentioning

confidence: 99%

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism

Scinto

Gupta

Thorat

et al. 2018

J Virol

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The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8 cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4 T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4 T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans. Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

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Section: Construction Of Infectious Proviruses and Generation Of Virumentioning

confidence: 99%

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism

Scinto

Gupta

Thorat

et al. 2018

J Virol

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“…However, some historical studies have been performed with viruses of uncertain provenance (e.g., unknown passage history). This is important because it is known that amplification of other filoviruses, including EBOV and Sudan virus (SUDV), results in genetic changes that impact pathogenesis in animal models ( 11 – 13 ). However, the effect of amplification of MARV on pathogenesis is unknown.…”

Section: Introductionmentioning

confidence: 99%

“…However, the effect of amplification of MARV on pathogenesis is unknown. Furthermore, one of the more notable changes found after cell culture passage of EBOV and SUDV occurs at the glycoprotein (GP) RNA editing site ( 11 , 12 ). However, MARV does not contain this editing site, so it is difficult to predict how cell culture passages might affect expression of GP in MARV.…”

Section: Introductionmentioning

confidence: 99%

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

Alfson

Avena

Delgado

et al. 2018

mSphere

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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 wellcharacterized 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 highpassage-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

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“…While it has been performed for SUDV, there are no published reports comparing the potential difference in pathogenesis between 7U and 8U EBOV in NHPs [ 7 ]. One may anecdotally compare what data are available for NHP experiments that were identified as using either 7U or 8U challenge stocks; however, this type of comparison is difficult due to the lack of sequence data, or viral pedigree, presented for historical studies.…”

Section: Discussionmentioning

confidence: 99%

“…Guinea pigs infected with the 8U EBOV also experienced a delay in death [ 6 ]. A comparison experiment between 7U and 8U has not been conducted in NHPs for EBOV, but Alfson et al, compared the two for SUDV and found no differences [ 7 ]. The two other mutations that arise in the EBOV viral population after passage in vitro were non-synonymous mutations at position 6179 (E47D) in the glycoprotein and at position 10833 (R163K) in VP24.…”

Section: Introductionmentioning

confidence: 99%

Ebola Virus Infections in Nonhuman Primates Are Temporally Influenced by Glycoprotein Poly-U Editing Site Populations in the Exposure Material

Trefry

Wollen

Nasar

et al. 2015

Viruses

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Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein’s poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations.

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

Cited by 14 publications

References 29 publications

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism

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

Ebola Virus Infections in Nonhuman Primates Are Temporally Influenced by Glycoprotein Poly-U Editing Site Populations in the Exposure Material

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