A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

Abstract

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

Mechanisms of Cell Entry by dsRNA Viruses: Insights for Efficient Delivery of dsRNA and Tools for Improved RNAi-Based Pest Control

“…A similar combination of cell line transfection with amplification in larvae was also used to construct a reverse genetics system for Dendrolimus punctatus cypovirus (DpCPV; Zhang et al, 2019 ). In this case, recombinant baculoviruses were employed to express T7 RNA polymerase in lepidopteran Sf9 cells which was used to drive expression of viral RNAs from co-transfected plasmids with T7 polymerase expression cassettes.…”

Biological Control Against Four-Stored Product Beetles Pests by Using Cytoplasmic Polyhedrosis Virus (Cypovirus1)

“…The highest percentage of mortality was recorded by concentrate 10/100 on tobacco beetle, drugstore beetle, darkling beetle larvae, and rice weevil adults (96.67%, 96.11%, 92.78%, and 90.00%) respectively, followed by concentrate 10/500 recorded 94.44% and concentrate 10/1000 recorded 70.56 %, on tobacco beetle larvae. On the other hand, a lower percentage of mortality was recorded by concentrate 1/25000 on the adult stage of rice weevil (33.33%), (Table 2), these results were illustrated with [14][15][16].…”

“…However, sudden destruction of the central gastric tissue leads to larvae abstaining from feeding, causing a series of physiological changes accompanied by symptoms of vomiting and diarrhea. To the death of the larva after 10 days of inoculation [14][15][16]. This work was carried out to viral identity was confirmed by host range studies.…”

Transcriptional Responses of Daphnis nerii Larval Midgut to Oral Infection by 2 Daphnis nerii cypovirus-23

“…In addition to Daphnis nerii, it has been found that DnCPV-23 can also induce infection and death in many species of Sphingidae insects, such as Cephonodes hylas Linnaeus, Ampelophaga rubiginosa Bremer & Grey, and Agathia lycaenaria Kollar. The genome of DnCPV-23 consists of ten segments of linear double-stranded RNA, referred to as Genomic Segments 1 (S1) to 10 (S10), in accordance with the fragments from longest to shortest [7]. Our previous research and unpublished data demonstrated that the virus can successfully replicate on the Sf9 [8] and Manduca sexta cell lines QB-MS 2-2 [9].…”