Virus and 4′-FlU
A lineage VII LASV isolate Germany ex Togo/2016/7082 (Genbank accessions KU961971 and KU961972) originated from serum of a LASV-infected patient who was medically evacuated to Germany from Togo59. The study challenge material was from the third passage of Vero 76 cells (ATCC CRL-1587) exposed to this serum. Authentication of Vero 76 cells was not performed beyond that performed by the providing repository (American Type Culture Collection (ATCC)). The passage two isolate was from the European Virus Archive and was obtained from T. Rieger and S. Gunther and passaged once at University of Texas Medical Branch (UTMB). The cell supernatants were stored at −80 °C as aliquots of ~1 ml. No mycoplasma or endotoxin was detected (<0.5 endotoxin units (EU) ml−1). 4′-FlU was obtained from MedChemExpress (HY-146246) and prepared in DMSO as 60 mM working stocks and frozen at −80 °C until use.
Study oversight
All study protocols described were approved by the UTMB Institutional Animal Care and Use Committee (IACUC) which were compliant with UTMB Institutional Biosafety Committee (IBC) guidelines under BSL-4 containment. UTMB animal facilities used in this work are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adhere to principles specified in the eighth edition of the Guide for the Care and Use of Laboratory Animals, National Research Council.
NHP challenge and treatment
Prior to conducting the 4′-FIU study, a power analysis was performed to determine the minimum cohort size for the study. For LASV, assuming a one-tailed alpha of 0.05, sample sizes of 5 per group will provide >80% power to detect a difference in the proportion of surviving animals between the treatment group (100% survival rate) and the control group (0% survival rate), using a Fisher’s exact test. For the 4′-FIU treatment study, animals were assigned to treatment or control groups using a random number generator in Microsoft Excel. All animals were anaesthetized via intramuscular injection with ketamine (10 mg kg−1) prior to procedures (for example, blood collection, weight and rectal temperature measurement). An initial study of 5 (2 male, 3 female) healthy adult AGMs (C. aethiops, PreLabs) weighing ~3.3 to 6.1 kg was performed to determine the pathogenic potential of LASV Togo in AGMs. All five monkeys were challenged by intramuscular injection in the left quadricep with a target dose of 1,000 PFU of LASV Togo (actual dose 840 PFU). As all five of these LASV Togo-infected AGMs succumbed to Lassa fever, a single control animal was used to confirm lethality of the challenge material used in the treatment study in which clinical parameters were compared with the five AGMs derived from the initial model experiment using the identical challenge material (that is, same virus passage). This allows for an ethical reduction in the number of animals used for data with highly predictable, lethal outcomes.
For the 4′-FIU treatment study, 6 (2 male, 4 female) healthy adult AGMs (PreLabs) weighing ~2.9–6.3 kg were challenged by intramuscular injection in the left quadricep with target dose of 1,000 PFU of LASV Togo (actual dose 1,312 PFU). Assignment to the treatment group or untreated positive control group was determined prior to challenge by randomization by Excel. Blinding was not performed for this study. Five monkeys were treated by oral gavage with 5 mg kg−1 4′-FIU (as a 1:10 suspension of 4′-FIU in DMSO to 1% methyl cellulose in water vehicle) beginning 6 days after LASV-Togo exposure. These 5 monkeys received daily doses of 4′-FIU for 10 days (6–15 DPI). The LASV Togo positive control monkey was not treated. The duration of the study was 35 days. This study was not blinded. All six AGMs were monitored daily and scored for disease progression with an internal LASV humane end-point scoring sheet approved by the UTMB Institutional Animal Care and Use Committee. The scoring changes measured from baseline included posture and activity level, attitude and behaviour, food intake, respiration, and disease manifestations, such as visible rash, haemorrhage, ecchymosis or flushed skin. A score of ≥9 indicated that an animal met the criteria for euthanasia.
Haematology and serum biochemistry
Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, haematocrit values, total haemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular haemoglobin concentrations were analysed from blood collected in tubes containing EDTA using a Vetscan HM5 laser-based haematologic analyser (Zoetis). Serum samples from blood collected in serum-separating tubes were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, calcium, creatinine, C-reactive protein, γ-glutamyltransferase, glucose, total protein and uric acid by using a Piccolo point-of-care analyser and Biochemistry Panel Plus analyser discs (Abaxis).
RNA isolation from LASV-infected AGM
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