Spliced mRNAs detected during the life cycle of Chicken anemia virus

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Abstract
The existence of spliced mRNA in Chicken anemia virus (CAV) was investigated, as three proteins appeared to be derived from a single 2.0 kb mRNA species. Human Torque teno virus (TTV), which displays a number of genomic similarities to CAV, is known to transcribe three mRNA species, suggesting that CAV may also have multiple mRNAs. Northern analysis of infected chicken MDCC-MSB1 cells revealed a 2.0 kb mRNA 3 h post-infection (p.i.) and additional 1.6, 1.3 and 1.2 kb bands visible at 48 and 72 h p.i. MDCC-MSB1 or COS1 cells transfected with a CAV clone showed similar results. The poly(A)+ RNA of infected cells was subjected to RT-PCR using a suite of CAV-specific primers. The major 2.0 kb RNA reacted with every primer, but the 1.3 and 1.2 kb RNAs only annealed to certain primers. The 2.0 kb mRNA had no deletions or mutations and was capable of encoding all three known CAV proteins. The 1.3 kb RNA had a splice site joining nt 1222 to nt 1814 and encoded head/tail viral protein 1 (VP1) without a frameshift. In addition, the 1.2 kb RNA possessed a splice site joining nt 994 to nt 1095 and encoded several putative, novel proteins with frameshift mutations. These splice sites conformed to the previously described GT–AG splicing rule. One further 0.8 kb RNA species appeared to be derived from a homologous recombination event. Discovery of the presence of spliced mRNA in CAV strengthens the similarity between CAV and TTV.