The sequence of LZ22 complete genome has been deposited in GenBank (Access Number, FJ455842). And the PIV3 complete genome sequence alignment dataset was analyzed employing RDP3 software package for scanning the recombinant sequence. And the isolate LZ22 was found to have greatly strong recombination signal: RDP, p-value < 10-21; GENECOVY, p-value < 10-21, Bootscan, p-value < 10-20, MaxChi p-value < 10-3; Chimaera p-value < 10-7; Siscan, p-value < 10-8, 3Seq, p-value < 10-13. And a breakpoint was located at position 485. Two strains GP and ZHYMgz01 were suggested as representatives of its putative parent lineages.
And then, Simplot software package was used to determine the recombination event [15
]. Employing the Findsites subprogram of SimPlot, one potential breakpoint was located at parsimonious regions with the maximization of χ2
, from positions 485 to 615 (χ2
= 122.3, P
< 0.0001 of Fisher's exact test). A similarity plot (Figure ) which was constructed by using all sites, revealed that the sequence of LZ22 showed greater affinity with one putative parent lineage of GP in the region from position 1 to 485 than the other putative parent ZHYMgz01 (100% versus 94%). However, sequence from positions 486 to 15536, ZHYMgz01 shared greater similarity with LZ22 than GP (98% versus 95%). P
value (Fisher's Exact Test) and χ2
value of the breakpoint were shown on the vertical line in Figure . The identical evidence also appeared in BootScanning result (Figure ). The region from GP lineage spanned the amino terminal 1/3 of the N protein approximately.
Figure 1 (A, B) Results of Similarity and BootScanning analysis of the genome of LZ22_FJ455842. The y-axis in Similarity plot (A) gives the percentage of sequence identity within a sliding window of 400 bp wide centered on the position plotted, with a step size (more ...)
At last, The phylogenic trees were also constructed using Mega 4 to determine the recombination events [16
]. From positions 1 to 485, LZ22 and GP were clustered into the same sublineage with 98% bootstrap value, while ZHYMgz01 was grouped into distinct sublineage (Figure ). But, in the other portion, the arrangement of the phylogenetic tree reflecting the relationship of the three isolates was in contrast with the previous one (Figure ). The topology of the two phylogenetic trees around the breakpoint showed a significant statistic discrepancy when the mosaic was included in analyzed data (Shimodaira-Hasegawa test, P < 0.001), constituting a powerful evidence for recombination.
Figure 2 Phylogenetic profiles of separate regions of LZ22_FJ455842 partitioned by cross-over events. The scale corresponds to the number of nucleotide substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in (more ...)
The minor putative parent of LZ22 was isolated from Japan, suggesting the existence of a global reservoir of HPIV with local subreservoirs supporting extensive levels of virus circulation, which permitted co-infection and resulted in the recombination event at last. The potential breakpoint 485 presents in gene N coding nucleoprotein spanning the region from positions 56 to 1737 of genomic sequence [17
]. In the previous study, viable artificial chimeric HPIV3 recombinants were constructed, which contained the nucleoprotein open reading frame (ORF) from either BPIV3 Kansas or shipping fever (SF) strain in place of the HPIV3 N ORF. The artificial recombinant (PIV3) in which the nucleocapsid N protein had been replaced by that of bovine PIV3 was found to be attenuated in primates [18
]. Here, we isolated a HPIV3 with a natural mosaic in NP ORF. Interestingly, before the breakpoint, the similarity of peptide sequences was up to 98.5% (129/131) although the similarity of gene sequences was only 94% between the two putative parent lineages. It is unknown whether the recombination in N gene is also associated with their adaptation in host cell via a changed virulence.
In addition, since there has been no report to show homologous recombination can take place between PIVs before this study; we analyzed 55 isolates (Table ) from GenBank in order to determine to what degree genetic diversity of the virus is affected by the homologous recombination. Five additional mosaic PIV sequences were detected through the analysis of sequences from isolates characterized in previous reports:, HT88 (U01082
], HT89a [19
), HT89c (U01085
], 81-19252_Texas-81 (EU439429
] and 92-7783_ISU-92 (EU439428
] (Table ). Two HN gene mosaics, HT88 and HT89a shared the same recombination event (Table ), suggesting they descended from the same mosaic ancestor. Please also refer additional files 1
, and 4
for the detail recombination information of each mosaic strain. These results suggested that homologous recombination did play a potential role in the evolution of the virus.
Typical PIV isolates used in the study
Characteristics of PIV intragenic recombinants
Interestingly, two mosaic viruses 81-19252_Texas-81 (U439429) and 92-7783_ISU-92 (EU439428) were reported to be involved in a cross infection between swine and bovine [13
]. Both of the isolates had a mosaic L gene. The transcription and replication functions of the parainfluenza virus are associated with the large RNA polymerase protein. Additionally, polyadenylation, and RNA editing activities have to do with L protein [20
]. The two putative mosaics were isolated from pigs in the United States [13
] while both of their putative parent lineages (Shipping fever and 910N lineages) belonged to BPIV3. Viruses are largely species-specific with respect to their host and usually do not cross species boundaries [5
]. Recombination processes will allow some viruses to acquire many of the key adaptive mutations in a single step, and thus make a major leap in fitness, which might result in a change of host tropism [21
]. It might be necessary to further study whether the recombination event is relative to the BPIV3 cross-species infection.
In conclusion, this study provides the potential evidence that there is mosaic PIV in the field. Our observations show that homologous recombination is a molecular mechanism of PIV genetic diversity and evolution. Therefore, this study might be important for knowing the genetic basis resulting in the rapid change of PIV biologic characteristics.