Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3'-terminal sequence in genome replication

To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3' terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3' terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3' terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3'-terminal deletions appeared to have a restored 3'-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template.     

An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses

Summary The genome of the non-cardiovirulent coxsackievirus B3 (CVB3) strain CVB3/0 was cloned and sequenced to aid in the elucidation of the viral genetic basis for the CVB3 cardiovirulent phenotype. Reverse-transcribed sub-genomic complementary DNA (cDNA) fragments were enzymatically amplified using generic oligonucleotide primers and were assembled as a complete infectious genomic copy (pCVB3-0) downstream of the T7 RNA polymerase promoter. Positive-strand viral RNA transcribed from pCVB3-0 using T7 RNA polymerase and transfected into HeLa cells produced infectious virus (CVB3/0c). No differences in phenotype were observed comparing growth of CVB3/0c to the parental CVB3/0 in HeLa single-step growth curves, virus yields, or plaque size. When inoculated into C3H/HeJ mice. CVB3/0c achieved cardiac titers equivalent to the parental CVB3/0 and like the parental virus, demonstrated a non-cardiovirulent phenotype. The nucleotide sequence of the cloned CVB3/0 genome was determined and compared to the genomes of infectious cDNA clones of cardiovirulent CVB3 strains. Two consistent differences among nucleotides in non-translated regions and 8 amino acid differences relative to two well-characterized infectious cDNA copies of genomes from cardiovirulent CVB3 strains were identified.     

Efficient propagation of betanodavirus in a murine astrocytoma cell line

Betanodavirus, a bipartite RNA virus of fishes and a member of Nodaviridae family, targets nervous tissues and is the causative agent of viral nervous necrosis in marine farmed fish. Betanodavirus is thought to be propagated only in fish cells because betanodavirus has only been isolated in fish and it is not well propagated in mammalian culture cells. However, the host specificity of betanodavirus has not yet been well analyzed. To analyze the host specificity of betanodavirus, various mammalian cells were screened for their permissiveness to betanodavirus. As a result, redspotted grouper nervous necrosis virus can be propagated efficiently in the murine astrocytoma cell line, DBT. The level of viral production in DBT was 10-fold-higher than in the fish cell line, E-11. This result is the first to demonstrate the efficient propagation of betanodavirus in mammalian cells and may help to elucidate the mechanism of the host specificity of betanodavirus.     

Potential for the replication of the betanodavirus redspotted grouper nervous necrosis virus in human cell lines

Summary The determination of the host ranges of viruses is important because of the possible emergence of infectious agents, which may result from the zoonotic transmission of animal viruses to humans. The family Nodaviridae, whose members are non-enveloped, positive-stranded bipartite RNA viruses, is comprised of the genera Alphanodavirus and Betanodavirus, whose members predominantly infect insects and fish, respectively. The alphanodaviruses can also infect suckling mice and suckling hamsters, resulting in paralysis and death. Pigs near the site of isolation of the Nodamura virus (NoV), an alphanodavirus, have been reported to have high levels of NoV neutralizing antibody, suggesting that they may be part of the natural host range of this virus. Betanodaviruses are the causative agents of viral nervous necrosis, which occurs in several species of fish. However, little is known regarding the mechanism of infection of these viruses. Whether betanodaviruses can infect hosts other than fish remains unclear. In this study, we examined the possibility that a betanodavirus, redspotted grouper nervous necrosis virus (RGNNV), can infect human cell lines and showed that this virus can attach to the cells but cannot penetrate them, although human cells can support the replication of the betanodavirus when viral RNAs are transfected. The betanodavirus in its present form cannot infect human cells. Correspondence: Nobuyuki Kobayashi, Laboratory of Molecular Biology of Infectious Agents, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan