Population dynamics of rhesus macaques and associated foamy virus in Bangladesh

Figures & data

Figure 1 Rhesus macaque sampling site locations including relative geographic proximity and historical and extant forest patches. Rhesus macaques, Macaca mulatta, were sampled from six geographically distinct sites in Bangladesh (n=126). Concentric circles in 10 km intervals from the epicenter of the sites are shown. Existing forest areas are noted in dark green and historical forest cover is shown in lighter green. Natural macaque migration would have been possible between sites that were connected by forested areas. Nomadic Bade people travel throughout the subcontinent with their performing macaques. We sampled 38 of these performing monkeys from multiple locations within the shaded light blue area.

Table 1 Each area, estimated population size and number of monkeys sampled by age class and sex

Location	Dokhola	Bormi	Dhamrai	Narayanganj	Charmaguria	Karamjal	Performing
Years sampled	2011	2007; 2009; 2011	2006; 2011	2010; 2011	2008; 2009	2011	2006; 2008; 2010; 2011
Context	Forest reserve	Urban	Urban	Urban	Urban	Forest reserve	Performing
Estimated population size^a	>300	102	102	55	210	>300	500–5000^a
Sample size	17	29	21	19	28	12	38
Male; female	8; 9	17; 12	9; 12	12; 7	9; 19	6; 6	15; 23
Juveniles; adults^b	0; 17	3; 26	4; 17	1; 18	3; 25	0; 12	5; 33

^aPopulation surveys in the urban and forested areas as reported by Hasan et al.²⁸ Estimates of performing monkey numbers are based on interviews with community leaders.

^bAnimals were considered Juveniles if based on their dental eruption sequence they were less than 24 months old.

Figure 2 Relationship between monkey and viral population structures. Each animal is represented by a single column spanning the figure’s sections. Columns are sorted by viral diversity, animal age, and sampling population. (A) Normalized Hamming distance for each pair of clones from each animal, representing intra-host viral diversity. The dotted line represents expected distance (due to PCR error) between distinct PCR products of a single DNA molecule. (B) The identifier of the animal to which each column corresponds. Respectively, (C), (D) and (E) represent number of viral clones, animal age class and sampling population for each monkey. (F) Microsatellite clusters as inferred by STRUCTURE; admixture between genotype clusters is represented by bar height. Microsatellite colors are assigned according to an observed correlation with sampling location. (G) Viral strains cloned from each animal, classified as described in the text. All sampled monkeys are represented except for one FV negative infant monkey (MBG211).

Figure 3 Foamy virus sequence diversity overview. Phylogenetic tree built from all sequenced gag nucleotide clones using FastTree using the Jukes–Cantor sequence evolution model. Due to recombination, this tree should not be interpreted as an evolutionary history, but rather as an indication of the clustering seen in the sequence data. The edges of the tree are labeled with SH-like local supports.

Table 2 Assay of anti-Gag antibodies using ELISA

Dilution	1∶50^a	1∶100^b	1∶200^b	1∶400^b	1∶800^b	Total
Reactivity	14	1	1	5	125	146

^aPlasma are considered negative if they had an OD450 nm reading less than one at this dilution.

^bThe highest dilution where an individual MBG plasma was reactive to Gag antigen.

Table 3 Geographic and age distribution of SFV seropositive rhesus macaques

	Infant^a	Juveniles^b	Adults^c	Total
Dhamrai	NA	1/5	16/16	17/21
Bormi	NA	8/8	21/21	29/29
Charmaguria	0/1	11/12	15/15	26/28
Narayanganj	NA	4/5	14/14	18/19
Karamjal	NA	2/2	10/10	12/12
Dokhola	NA	4/4	13/13	17/17
Performing	NA	4/5	26/33	30/38

^aInfants: animals aged less than 9 months based on dental formula.

^bJuveniles: animals aged at less than 24 months based on dental eruption sequence.

^cAdult: animals with erupted first and second molars and permanent anterior dentition.

NA: no animal in this group were sampled.

Figure 4 Splits network giving an overview of sequence diversity, and built by SplitsTree with strain labels. SplitsTree produces a ‘splits network’ capable of displaying non-treelike sequence difference information in a graphical form. In the figure, each set of parallel edges represents a ‘split’, which is a collection of sequence differences that separates one group of sequences from another. In this case, non-tree-like evolution is expected to be from recombination; putative recombinant sequences are found at the end of the parallelograms formed by edges. Strain labels have multiple identifiers if they were found to be recombinant by cBrother; e.g., a/b indicates a recombinant between core sequences a and b. An underscore (_) indicates the presence of a >200 bp region in which no one core strain was clearly found as the source.

Figure 5 Amino-acid alignment of ‘Core’ strain representatives. Translated Gag sequence alignment between strain representatives compressed to variable sites. The numbers on the top of the figure show the position of these non-constant columns relative to the beginning of our gag sequencing region: the top digit is the 100’s place, the middle the 10’s place, and the bottom is the 1’s place.

Figure 6 Recombination between Bormi strains and non-recombinant parental strains. Phylogenetic trees constructed by running PhyML on representative sequences which have been partitioned into separate alignments along recombination breakpoints inferred by cBrother. (A) and (B) show the relationship between Bormi1 and the core strains; (C) and (D) the relationship between Bormi2 and the core strains. (A) is built from sequence data between nucleotides 1 and 304, (B) from 305 on, (C) from 1 to 496 and (D) from 497 on (nucleotide (nt) number refers to position in the 1125 nt fragment of gag we sequenced, not to the nt position of the full length gag gene). Internal branches are labeled with their bootstrap values to indicate confidence.

Table 4 Percentage of amino-acid sequence similarity^a in the region of gag analyzed between strains and SFV-1

	Bormi1	Bormi2	Charmaguria	Dhamrai	Dokhola	Karamjal	SFV-1
Bormi1		99	99	99	99	97	95
Bormi2			99	99	99	97	95
Charmaguria				99	99	95	95
Dhamrai					99	96	95
Dokhola						97	95
Karamjal							93

^aSimilarity between two strains is expressed as percentage of identical amino acids and conservative substitutions. The amino-acid sequence is derived from a consensus of all clones for each strain, using the portion of gag that we sequenced (1235 nt). SFV-1 is the cloned macaque SFV described by Mergia et al.³⁶

Table 5 Infectivity of SFV-1 with a 6 nucleotide deletion in gag

Virus^a	Titer^b (units/mL)
WT SFV-1	2.0×10^5
pSFVΔ6nt-1	9.5×10^4
pSFVΔ6nt-2	1.2×10^4
pSFVΔ6nt-3	3.0×10^6

^aWT SFV-1 or 3 randomly picked molecular clones containing the 6 nucleotide deletion in gag were transfected into Tf cells. After 48 h, supernatants were collected.

^bSupernatants were titered on simian foamy-activated beta-glactosidase cells.

Hasan MK, Aziz MA, Alam SMR et al.Distribution of rhesus macaques (Macaca mulatta) in Bangladesh: inter-population variation in group size and composition. Primate Conserv2013;26: 125–132.

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