Infectious rabies viruses from cloned cDNA.

Abstract

The generation of infectious rabies virus (RV), a non‐segmented negative‐stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full‐length RV antigenome‐like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non‐translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non‐essential genetic material might be present in the genomes of non‐segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure‐‐potentially applicable also for other negative‐stranded viruses‐‐greatly facilitates the investigation of RV genetics, virus‐host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

“…In contrast to previous attempts with SeV and other mononegaviruses Garcin et al 1995;Lawson et al 1994;Radecke et al 1995;Schnell et al 1994;Whelan et al 1995), our system consistently recovered infectious virus even from pSeV(ÿ) that generates (ÿ)RNA (Table 1). Although the frequency was lower than that from pSeV( ) (1/50-1/100), an additional passage in eggs was sufficient to generate maximal amounts of virus (Table 1).…”

“…The antisense effect of mRNAs on the (ÿ)RNA supposedly explains the difficulties involved in recovering virus from (ÿ)RNA-specifying cDNA (Schnell et al 1994;Lawson et al 1995;Garcin et al 1995). This antisense problem may also explain our 50-to 100-fold less efficient virus recovery from pSeV(ÿ).…”

“…The recovery of infectious viruses from cloned and transfected cDNA will be an innovative technology, enabling the genetic engineering of viruses in this superfamily. The DNA transfection system has been established for two rhabdoviruses: rabies virus (Schnell et al 1994) and vesicular stomatitis virus (VSV) (Lawson et al 1995;Whelan et al 1995), with a genome of about 12 kilobases (kb), and for three paramyxoviruses: measles (Radecke et al 1995), Sendai (Garcin et al 1995), and RS (Collins et al 1995) viruses, with an approximate 15 kb genome. These systems, except for that of the measles virus, employed the transfection of cDNA into cells in which the cDNA and the plasmids carrying the viral nucleocapsid protein (NP or N) and RNA polymerase genes are coexpressed by vaccinia virus (VV)-driven bacteriophage T7 RNA polymerase (T7 pol).…”

“…Starting with genomic negative sense (ÿ)RNA was unsuccessful. The reason for this seems to be that unlike ( )RNA, (ÿ)RNA presumably hybridizes to the viral mRNAs supplied in trans (Schnell et al 1994;Lawson et al 1995;Garcin et al 1995), or that T7 pol aberrantly terminates transcription that generates (ÿ)RNA (Whelan et al 1995). In addition, infectious virus recovery is quite rare, since sometimes only one infectious unit is obtained from about 10 7 or even more transfected cells (Schell et al 1994;Lawson et al 1995;Garcin et al 1995).…”

Rescue of Mumps Virus from cDNA

“…Only the intact nucleocapsid structure can act as template for RNA transcription, replication, and subsequent virus amplification; therein lies the difficulty in genetic manipulation of MUV and other negative-strand RNA viruses. Unlike the positive-strand RNA viruses, where naked genomic RNA is infectious and infectious virus can be recovered from a cDNA copy of the genome in the absence of additional viral factors (31,39), the naked genome of nonsegmented negative-strand RNA viruses is not infectious, and rescue of virus from cDNA requires at least intracellular coexpression of viral NP, P, and L proteins, along with a full-length positive-sense genome RNA transcript, all under control of a bacteriophage T7 RNA polymerase promoter (2,3,6,9,14,15,16,19,29,32,33,40). In all of the rescue systems described so far for the nonsegmented RNA viruses, T7 RNA polymerase was supplied either by a coinfecting recombinant vaccinia virus (12,41) or by endogenous expression in a transformed cell line (32).…”