Abstract
Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.
Highlights
Schistosomiasis is considered the most important helminthic disease of humanity in terms of morbidity and mortality, and is one of the major neglected tropical diseases (NTDs) [1,2,3,4]
Using a lab-modified form of human immunodeficiency virus-1 (HIV-1), we manipulated the genome of Schistosoma mansoni, one of the major species of schistosomes
The findings were notable since they revealed that HIV-1 was active within cells of S. mansoni, and they provide the first demonstration that HIV-1 can integrate into the genome of an invertebrate
Summary
Schistosomiasis is considered the most important helminthic disease of humanity in terms of morbidity and mortality, and is one of the major neglected tropical diseases (NTDs) [1,2,3,4]. To accelerate discovery of intervention targets for schistosomiasis and to provide exploitable insights into the parasite biology and pathogenesis, concerted efforts are in train to produce reference genome sequences of the human schistosomes and related helminths [6,7,8,9,10,11]. Because parasitic flatworms at large are difficult to maintain in the laboratory due to complex developmental cycles, they remain recalcitrant to genetic/cellular manipulations, presenting a significant bottleneck for adapting state-of-the-art approaches to elucidate gene function [12]. Current large-scale approaches, mainly involving medium-throughput RNAi screening [13, 14], currently provide a veneer only of information on gene function since the knowledge of characteristics and regulation of specific gene expression remains limited. To profoundly probe function at scale, protocols for routine manipulation of the genome need to be established and optimized; genes need to be disrupted, transgenes inserted, and expressed in a sustainable, and even tunable, fashion
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