Saimirus

Estimation of the total body parasite burden from plasma PfHRP2 has been described in detail in Asian adults with severe malaria and requires incorporation of an elimination half-life estimate [15] (link). This was assessed separately in African children because clearance might be dependent on immunity (antibodies against PfHRP2), which has a higher level in high transmission settings, and PfHRP2 production is parasite strain dependent [20] (link). Plasma PfHRP2 half-life was assessed in 30 patients from Tanzania from samples taken on admission and after 3 and 7 days following treatment. Separate ethical approval for this sub-study was obtained from the Ethics Committee of the National Institute for Medical Research, Tanzania. These data were analysed using WinNonlin statistical package (Pharsight, Mountain View, California, US). Individual PfHRP2 concentration-time curves were fitted according to a first-order elimination model. From this, a mean (95%CI) plasma elimination half-life (t_½) was estimated as 1.10 (0.91 to 1.29) days, or 0.55 erythrocytic cycles. Half-life was not significantly different between treatment arms, and was not correlated with renal function (estimated by blood urea nitrogen [BUN]). A parasite multiplication factor of 3 immediately before peak parasitaemia was assumed, based on in-vitro and Saimiri monkey studies of African parasite strains causing severe malaria [21] (link),[22] (link). Higher multiplication rates were explored in a sensitivity analysis [23] (link),[24] (link). The formula for total parasite burden is: P_tot = 7.3×PfHRP2×(1−Hct)×body weight [kg] ×10¹³, with PfHRP2 in g/L [15] (link). The differences in the current formula with the one used earlier in adult Asian patients result from the different estimates for plasma PfHRP2 half-life and parasite multiplication rates. The circulating parasite burden was calculated from the peripheral blood: parasites/µl×10⁶×blood volume ( = 0.08×weight [kg]) [15] (link). The sequestration index was calculated as total parasite burden/circulating burden [25] (link).

Eleven laboratory-reared squirrel monkeys (Saimiri spp.) from a breeding colony of the National Primate Center (CENP) Ananindeua, Pará, Brazil were used in the study. Before the infection all animals were bled and tested negative by hemagglutination-inhibition (HI) test for the presence of antibodies against YFV, dengue virus 1–4, Ilheus virus, Rocio virus, and Sant Louis virus. The non-immune animals were intradermally infected with YFV genotype I isolate BeH655417 (infectious dose: 1 × 10⁶ plaque forming units (PFU)/mL). One animal was not infected and served as a negative control. The YFV sample used in this study was originally obtained from a severe fatal human case in Roraima, Brazil in 2014 and was genetically characterized as of South American genotype I (BeH655417) and isolated in the Evandro Chagas Institute (IEC), Ananindeua, Pará, Brazil and propagated by a single additional passage in C6/36 cells. Infection by YFV cells was confirmed by indirect immunofluorescence assay with polyclonal and monoclonal anti-YFV antibodies [8 (link)].
One monkey was euthanized each day during the first 7 days and at 10-, 20-, and 30-days post-infection (dpi). For necropsy, the animals were euthanized intravenously with ketamine (15 mg/kg) and xylazine (1 mg/kg) [9 ]. Subsequently, macroscopic examination and photographic documentation (Fujifilm S290, Tokyo, Japan) was performed. Liver tissue was collected as three individual samples: two were preserved at −70 °C and the third was fixed in 10% buffered formalin for 24 h and then stored in 70% ethanol until processing.

The RepeatMasker utility program COSEG was applied to the lineage-specific owl monkey and capuchin monkey Alu insertions to determine the subfamily composition based on co-segregating mutations. Alu insertions determined to be lineage-specific were aligned via Crossmatch (www.phrap.org/phredphrapconsed.html, accessed on 1 January 2023 with the default settings, then analyzed via COSEG (www.repeatmasker.org/COSEGDownload.html; accessed on 19 December 2022) to determine the subfamily structure. The dataset was aligned against the AluS consensus sequence [30 (link)]. COSEG was then used to group the Alu subfamilies. The middle A-rich region of the AluS consensus sequence was excluded from the analysis when determining the subfamilies, whereas tri and di segregating mutations were considered. A group of ten or more identical sequences was considered a separate Alu subfamily. The consensus sequences were subjected to a RepeatMasker analysis using 24 subfamilies previously defined by RepBase [15 (link),24 (link)], as well as the 86 from marmoset and 46 from squirrel monkey (see Section 1) to remove exact matches. Eliminating subfamilies duplicated in owl monkey or capuchin resulted in non-overlapping datasets. These 189 Alu subfamilies were then aligned in BioEdit [31 ] and a network analysis was completed based on the accumulation of diagnostic mutations.

Four high-quality platyrrhine genomes (common marmoset; C. jacchus [caljac3], capuchin monkey; Cebus imitator [Cebus_imitator-1.0], squirrel monkey; S. boliviensis [SaiBol1] and owl monkey; Aotus nancymaae [Anan_2.0]) were obtained from the National Center for Biotechnology Information (NCBI) and analyzed for their Alu content using RepeatMasker (RepeatMasker-Open-4.0). Ascertainment of lineage-specific or recently integrated Alu insertions from the owl monkey genome [Anan_2.0] and from the C. imitator genome [Cebus_imitator-1.0] [25 (link)] were performed as described previously [26 ,27 (link),28 (link),29 (link)]. Briefly, full-length Alu elements were extracted from the RepeatMasker output using a custom python script (described at link https://github.com/t-beck; accessed on 19 December 2022). These elements, along with 600 bp 5′ and 3′ flanking sequence, were then compared to the remaining genomes by means of a sequential BLAT [23 (link)] conducted in the following order: (1) human (Homo sapiens; [GRCh38.p13]); (2) common marmoset (C. jacchus; [caljac3]); (3) capuchin monkey (C. imitator; [Cebus_imitator-1.0] or owl monkey (A. nancymaae; [Anan_2.0] and (4) squirrel monkey (S. boliviensis; [SaiBol1.0]). A sequential BLAT involved analyzing the output after each BLAT for capuchin or owl monkey-specific Alu elements compared to the other four genomes.