Characterization and construction of functional cDNA clones of pariacoto virus, the first Alphanodavirus isolated outside Australasia

Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyw orm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and a bout 30 nm in diameter, The virus has a bipartite RNA genome and a single m ajor capsid protein with a molecular mass of 39.0 kDa, features that suppor t its classification as a Nodavirus. As such, PaV is the first Alphanodavir us to have been isolated from outside Australasia, Here we report that PaV replicates in was moth larvae and that PaV genomic RNAs replicate when tran sfected into cultured baby hamster kidney cells. The complete nucleotide se quences of both segments of the bipartite RNA genome were determined. The l arger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-am ino-acid open reading frame (ORF) encoding protein A, the viral contributio n to the RNA replicase, During replication, a 414-nucleotide long subgenomi c RNA (RNA3) is synthesized which is coterminal with the 3' end of RNA1, RN A3 contains a small ORF which could encode a protein of 90 amino acids simi lar to the B2 protein of other alphanodaviruses, RNA2 contains 1,311 nucleo tides and encodes the 401 amino acids of the capsid protein precursor a. Th e amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, th e best characterized of the alphanodaviruses, These and other sequence comp arisons indicate that PaV is evolutionarily the most distant of the alphano daviruses described to date, consistent with its novel geographic origin. A lthough the PaV capsid precursor is cleaved into the two mature capsid prot eins beta and gamma, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV, To facilitate th e investigation of PaV replication in cultured cells, me constructed plasmi ds that transcribed full-length PaV RNAs with authentic 5' and 3' termini, Transcription of these plasmids in cells recreated the replication of PaV R NA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral protei ns A and a.