Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: applications on detection, sequencing and virus isolation.
Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93-99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10(5.55) TCID(50)/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.
Dezen
Diogenes
D
Laboratório de Virologia, FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Caixa Postal 47, Eldorado do Sul, 92990-000 RS, Brazil. ddezen@gmail.com
Rijsewijk
Franciscus Antonius Maria
FA
Teixeira
Thais Fumaco
TF
Holz
Carine Lidiane
CL
Cibulski
Samuel Paulo
SP
Franco
Ana Cláudia
AC
Dellagostin
Odir Antonio
OA
Roehe
Paulo M
PM
D000818:Animals; D018173:Circoviridae Infections / Q000662:veterinary; D017925:Circovirus / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005784:Gene Amplification; D016679:Genome, Viral; D016133:Polymerase Chain Reaction / Q000379:methods; D013552:Swine; D013553:Swine Diseases / Q000821:virology; D014162:Transfection
D017931:DNA Primers; D004279:DNA, Viral