Recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed RNA

Abstract

We report the first full-length infectious clone of strain JX/CHA/97 of rabbit hemorrhagic disease virus (RHDV). The transcripts from the full-length cDNA clones were infectious when they were directly injected into rabbits. The sequence of the virus recovered from the rabbits was identical to that of the injected RNA transcripts. The cDNA clone was engineered to contain one silent nucleotide change to create an EcoRV site (A to T at nucleotide 2908). The genetic marker was retained in the recovered progeny virus. The transfection of RNA transcripts into RK-13 cells resulted in the synthesis of viral antigens, indicating that the cDNA clones were replication competent. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of RHDV.

FIG. 1.

Schematic diagram of steps used in the construction of a full-length cDNA clone of RHDV. At the 5′ end of the genome, the KpnI restriction site and an Sp6 RNA promoter were fused to the genome. The arrow indicates the transcription start site of Sp6 RNA polymerase. Downstream of the 3′ untranslated region, a poly(A) tail of 27 A's and the restriction sites NruI, NotI, and XbaI were inserted. The complete viral genome was divided into three fragments flanked by unique restriction sites, represented by the horizontal lines labeled AB, C, and D. The length of each fragment is indicated in parentheses. As shown at the bottom of the figure, fragments AB, C, and D were cloned into the pBluescript II SK+ vector in the order AB to D.

FIG. 2.

Transcription of RHDV RNA. Formaldehyde-denaturing 1.0% agarose electrophoresis of RNA transcript together with genomic RNA purified from RHDV.

FIG. 3.

IFA of viral protein expression in cells transfected with full-length RHDV RNA transcript. (Top) RK-13 cells transfected with in vitro transcripts. (Bottom) Normal RK-13 cells.

FIG. 4.

Immune electron micrograph of negatively stained RHDV particles.

FIG. 5.

Differentiation between cloned virus and parental JX/CHA/97 strain. An EcoRV restriction site was introduced in the full-length cDNA clone of JX/CHA/97 to allow discrimination between cloned virus (tagged with the EcoRV site) and parental virus (lacking an EcoRV site). RNA was extracted from the viral suspension of rabbits inoculated with either the cloned virus or the parental JX/CHA/97 isolate (lane A), and a 443-bp fragment was amplified by RT-PCR as described in Materials and Methods (lane B). The amplicons were digested with EcoRV and analyzed on a 2.0% agarose gel. The presence of an EcoRV restriction site resulted in fragments of 263 bp and 180 bp. As expected, the restriction site was found in the cloned virus but not in the parental JX/CHA/97 virus isolate. Molecular size markers (in base pairs) are noted at the right of blots.