What's in a strain? Viral metagenomics identifies genetic variation and contaminating circoviruses in laboratory isolates of pigeon paramyxovirus type 1

We used next generation sequencing on random amplified viral nucleic acids to determine the genome sequence of 11 pigeon paramyxovirus type 1 (PPMV-1) isolates from Belgium (period 1998–2011). The PPMV-1 deep sequence data allowed identification of sequence variability in multiple PPMV-1 isolates, including one STOP codon in the Matrix gene which was present in 15% of the viral population of one isolate. Notably, mutations that were previously associated with pathogenicity in chickens were identified as minor sequence variants in one parent laboratory strain. A phylogenetic analysis of the consensus PPMV-1 genome sequences was performed. In addition to providing nearly complete paramyxovirus genome sequences, our sequence-independent approach identified the presence of pigeon circovirus (PiCV) sequences in four of these viral stocks. Real-time quantitative RT-PCR analysis specific for PMV-1 and PiCV showed that these contaminations were present in seven viral stocks consisting of allantoic fluids and was occasionally also detected in stocks passaged in embryonated chicken eggs. Phylogenetic analysis of the PiCV consensus genome sequences showed a circulation of PiCV covering the full genetic diversity of known PiCV. This study shows the value of novel sequence independent technologies for access to sequence information for the control of reference virus stocks and other biological materials, as co-infecting viruses or sequence variants from the original sample may persist in the stocks without being identified by the routine virus-specific diagnostic tools. The exact role of PiCV in pigeon disease – in particular Newcastle disease – and its potential interference with PPMV-1 diagnostics remains to be investigated.