Synthetic transcripts of double-stranded Birnavirus genome are infectious.

Abstract

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented doublestranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Nonstructural Protein of Infectious Bursal Disease Virus Inhibits Apoptosis at the Early Stage of Virus Infection

“…This virus was plaque purified twice in secondary CEF cells and propagated in Vero cells. Primary CEF cells were prepared from 10-day-old embryonated eggs (SPAFAS, Inc., Storrs, Conn.) as described previously (33,46). Secondary CEF cells, maintained in a growth medium consisting of M199-F10 (50%-50% [vol/vol]) and 5% fetal bovine serum (FBS), were used in all experiments, including virus titration and plaque assays.…”

“…Poly(I · C) was prepared with nuclease-free water according to the instructions provided by the company (Amersham Biotech Inc.). CEF cells were transfected with 2 to 12 g of synthetic double-stranded RNA (dsRNA) [poly(I · C)], using Lipofectin reagent, as described previously (33,46). DNA fragmentation assay.…”

“…For this experiment, we recovered the recombinant rGLSA virus and its NS knockout mutant, rGLSNS⌬, using reverse genetics (29,33). When CEF cells were infected with these two viruses, no difference in viral protein synthesis (based on VP3 expression) was observed (Fig.…”

Extensive antigenic changes in an atypical isolate of very virulent infectious bursal disease virus and experimental clinical control of this virus with an antigenically classical live vaccine

“…Some nucleotide (nt) and the resulting amino acid (aa) changes occurring in segment A have been proposed as the basis for diagnostic tests for the putative vvIBDV, either on an antigenic basis (Eterradossi et al, 1997a,b) or on a molecular basis (Jackwood & Sommer, 1999;. However, recent progress in the reverse genetics of IBDV (Mundt & Vakharia, 1996) have allowed the demonstration that VP2 alone is not responsible for vvIBDV increased pathogenicity (Boot et al, 2000). Hence, it is not clear whether the conserved features among vvIBDV should be considered as pathogenicity markers, or merely as an indication that all vvIBDVs, which have now spread almost worldwide, North-America, Australia and New-Zealand excepted (for a review, see , may have a clonal origin.…”

Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus

“…Such reverse genetics systems allow artificial manipulation of viral genomes at the cDNA level by site-directed mutagenesis, deletion͞insertion, and rearrangement and have led to the accumulation of significant new knowledge relating to the replication, biological characteristics, and pathogeneses of these viral genera and families (5,9). For dsRNA viruses, which comprise three families, the Reoviridae, Birnaviridae, and Cystoviridae, such achievements have so far been restricted to the low-numbered segmented dsRNA viruses: two segmented birnaviruses (10,11) and three segmented 6 bacteriophage of the Cystoviridae (12). The Reoviridae viruses that possess 10-12 segmented genomes have been proven to be very refractory to this approach, except for the reoviruses.…”