Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis.
We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.
Jourdan
M
M
Station de Recherches de Pathologie Comparée INRA-UA CNRS 1184, Saint Christol-lez-Alès, France.
Jousset
F X
FX
Gervais
M
M
Skory
S
S
Bergoin
M
M
Dumas
B
B
D000818:Animals; D003001:Cloning, Molecular; D004267:DNA Viruses / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D007304:Insect Viruses / Q000235:genetics*; D009036:Moths; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D014764:Viral Proteins / Q000302:isolation & purification; D014771:Virion / Q000235:genetics*
D004279:DNA, Viral; D014764:Viral Proteins