Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.

Jana Hagen,Murray E. Selkirk,Peter Sarkies,Meera Goh Nair

doi:10.1371/journal.ppat.1009286

Abstract

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

Highlights

Nematode parasites are responsible for infection of large numbers of people and animals, and the latter issue represents a global agricultural problem which restricts economic growth in many countries [1]
We show here that foreign genetic material can be introduced into a widely used laboratory model of intestinal nematode infection by using a viral vector
The vector was modified to improve transgene expression, and a reporter protein expressed by transduced nematode larvae in vitro

Summary

Introduction

Nematode parasites are responsible for infection of large numbers of people and animals, and the latter issue represents a global agricultural problem which restricts economic growth in many countries [1]. No subunit vaccines for nematode infection have been commercialised, and multi-drug resistance to the major classes of anthelmintics is widespread in gastrointestinal parasites of livestock [2]. RNA interference in animals was discovered and characterised in Caenorhabditis elegans [3], it has proven extremely difficult to translate to animal-parasitic nematodes, i.e. those which infect humans, livestock and laboratory animal models. Systematic comparison indicates that most of the RNAi machinery is conserved across parasitic nematodes, with notable exception of the sid-2 gene, which encodes an intestinal luminal transmembrane protein required for environmental RNAi in C. elegans [7,8]. The major blockade to RNAi in most parasitic nematodes is likely to be effective delivery of sufficient dsRNA to trigger the silencing mechanism

Methods

Results

Discussion

Conclusion