Detection of Retroviral Super-Infection from Non-Invasive Samples

While much attention has been focused on the molecular epidemiology of retroviruses in wild primate populations, the correlated question of the frequency and nature of super-infection events, i.e., the simultaneous infection of the same individual host with several strains of the same virus, has remained largely neglected. In particular, methods possibly allowing the investigation of super-infection from samples collected non-invasively (such as faeces) have never been properly compared. Here, we fill in this gap by assessing the costs and benefits of end-point dilution PCR (EPD-PCR) and multiple bulk-PCR cloning, as applied to a case study focusing on simian foamy virus super-infection in wild chimpanzees (Pan troglodytes). We show that, although considered to be the gold standard, EPD-PCR can lead to massive consumption of biological material when only low copy numbers of the target are expected. This constitutes a serious drawback in a field in which rarity of biological material is a fundamental constraint. In addition, we demonstrate that EPD-PCR results (single/multiple infection; founder strains) can be well predicted from multiple bulk-PCR clone experiments, by applying simple statistical and network analyses to sequence alignments. We therefore recommend the implementation of the latter method when the focus is put on retroviral super-infection and only low retroviral loads are encountered

False negativity and positivity as a function of the number of bulk-PCR products analysed.

<p>(a) Probability to detect bimodality (<i>i.e.</i>, super-infection) in the frequency distribution of the number of mismatches per pair of sequences as a function of the number of bulk-PCR products analysed and for subjects for which EPD-PCR revealed a super-infection (n = 6); (b) Probability to detect bimodality in the frequency distribution of the number of mismatches per pair of sequences as a function of the number of bulk-PCR products analysed and for subjects for which EPD-PCR revealed a single infection (n = 4). Shown are median, quartiles, minimum and maximum, of the respective probabilities per subject.</p

Mismatch distribution analyses of EPD-PCR and clone sequence datasets.

<p>Individuals are ordered such that first four individuals with a single infection (as determined by EPD-PCR) are shown (B2, B3, B4 and T3; above the bar) followed by individuals with super-infections (below the bar). Plots are paired for each individual with the EPD-PCR dataset on the left and the bulk-PCR clone dataset on the right. EPD-PCR distributions are black; bulk-PCR clone distributions identified as unimodal (ΔAICc<2) are green; bulk-PCR clone distributions identified as bimodal are purple (ΔAICc>2).</p

Median joining network of bulk PCR product clone sequences.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036570#pone-0036570-g001" target="_blank">Figure 1</a>, the networks are ordered by infection status. Within each network, node size is proportional to the frequency of sequence occurrence (total n = 25 for each individual). Branch lengths are directly related to the number of mutations between sequences, with values noted for differences greater than two base pairs. Clone haplotypes a–f (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036570#pone-0036570-t001" target="_blank">Table 1</a>) are noted within or adjacent to their corresponding node. Networks generated using TCS were highly similar (data not shown).</p

Alignment of all clone sequences generated in the course of this study

FASTA file comprising 915 SFV int sequences

Retailing Under the N.R.A. II

Data are missing on the diversity of Plasmodium spp. infecting apes that live in their natural habitat, with limited possibility of human-mosquito-ape exchange. We surveyed Plasmodium spp. diversity in wild chimpanzees living in an undisturbed tropical rainforest habitat and found 5 species: P. malariae, P. vivax, P. ovale, P. reichenowi, and P. gaboni