Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for .50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (.75% survival rate as compared to ,15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture. R NA interference (RNAi) is a mechanism by which cells silence the expression of foreign genes. This process often provides an adaptive innate immunity against viruses where double-stranded RNAs encoded by the viruses during infection act as pathogen trigger after they are taken up by the cellular RNAi machinery (Wang et al. 2006). Alternatively, this natural defense mechanism is exploited as an antiviral therapy via the artificial inhibition of the expression of essential viral genes (Leonard and Schaffer 2006). Multiple protocols of delivery of dsRNA or of constructs encoding dsRNAs in the organism are currently under assay to combat infections of various viruses. The efficiency of the assays is still challenged by the delicate setting of the proper dosage of the RNAi, the relative longevity of the effect, the occurrence of RNAi driven toxicity, and the virus-intrinsic susceptibility. A few attempts have been made in animal and plant models where the antiviral trait was installed by transgenesis to confer stable protection to the transformed individuals and to their progeny. Successes have been reached in plants ( Bucher et al. 2006;Bonfim et al. 2007;Zhang 2010) but, to our knowledge, no case has yet been reported in animals showing a stable and robust protection against a virus after a RNAi-aided antiviral trait was introduced through germline transformation.The baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), is a major pathogen that affects silkworm rearings and hampers silk cocoon production in Asia. In India alone .50% of silk cocoon crop losses are attributed to baculovirus infection (Khurad et al. 2006). Effective treatment against the virus has been elusive due to its sturdy nature and the lack of control strategies. Interestingly, the biology of the virus is reasonably...

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus

“…Although one target site for the ie-1 gene abolished genomic cleavage because of a single-nucleotide mismatch between the gRNA template and its BmNPV genome target, our data showed that the remaining three target sites were sufficient to introduce large-segment deletions into BmNPV genomes and the relative abundance of BmNPV was decreased to a nearly undetectable level (>10 4 -fold) in TG silkworms (Fig. 4), which was quite different from the limited viral blockage (2.5- to ∼16.7-fold) achieved by RNAi-based antiviral strategies (8, 9). The markedly enhanced survival of the engineered silkworms supports the BmNPV-specific CRISPR/Cas9 system as a promising tool for application in sericulture.…”

“…Total DNA was extracted from silkworms treated with 10 6 OBs/larva in the TG-A, TG-B, TG-C, and WT groups every 12 h (from 0 to 72 hpi). Several BmNPV genes, including lef-1 , lef-3 , gp41 , and gp64 , were used in previous studies for viral DNA quantification (8, 9, 28). Among them, the lef-3 and gp64 genes, two essential genes beyond the range of the gRNA target deletion sequences, were selected as the detection indicators in quantitative PCR (qPCR) analyses of the relative abundance of BmNPV with Bmrp49 as the internal reference gene.…”

“…Only two antiviral genes, the endogenous antiviral gene Bmlipase- 1 and the exogenous antiviral gene hycu-ep32 , have been reported to enhance the resistance of silkworms to BmNPV infection (3, 4). In contrast, TG RNAi has been applied extensively by silencing one or multiple essential BmNPV genes to destroy specific viral mRNAs (7, 8). It is important to select appropriate genes as targets in RNAi-based anti-BmNPV experiments, as silencing of different BmNPV genes results in variable antiviral resistance efficiency (9).…”

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

“…piggyBac gene vectors are able to carry large fragments of exogenous DNA into genomes. In B. mori, the largest fragment integrated using a piggyBac vector was 12.4 kb [18]. Ma et al [13] reported that fragments 2-8 kb in length had no significant effect on the transformation efficiency of B. mori using piggyBac vectors.…”

The Genetics of Immunity

“…In contrast, agricultural systems can sometimes be engineered for disease resistance, as Subbaiah et al (2013) have shown by establishing stably transformed silkworms that are resistant to Bombyx mori nucleopolyhedrovirus (BmNPV). The transformed silkworms carry RNAi constructs that target four essential viral genes, generating .75% survival of experimental viral infection relative to ,15% survival in the parental strain.…”