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Detection and characterization of two co-infection variant strains of avian orthoreovirus (ARV) in young layer chickens using next-generation sequencing (NGS).

Tang Y, Lin L, Sebastian A, Lu H - Sci Rep (2016)

Bottom Line: The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins.Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains.Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains.

View Article: PubMed Central - PubMed

Affiliation: Wiley Lab/Avian Virology, Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, United States.

ABSTRACT
Using next-generation sequencing (NGS) for full genomic characterization studies of the newly emerging avian orthoreovirus (ARV) field strains isolated in Pennsylvania poultry, we identified two co-infection ARV variant strains from one ARV isolate obtained from ARV-affected young layer chickens. The de novo assembly of the ARV reads generated 19 contigs of two different ARV variant strains according to 10 genome segments of each ARV strain. The two variants had the same M2 segment. The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains. Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains. These findings have demonstrated the first naturally occurring co-infection of two ARV variants in commercial young layer chickens, providing scientific evidence that multiple ARV strains can be simultaneously present in one host species of chickens.

No MeSH data available.


Related in: MedlinePlus

Circos plot descriptions of the complete genomes of the two co-infection variant strains.(A) Reo/PA/Layer/01224a/14 variant (or PA01224a); (B) Reo/PA/Layer/01224b/14 variant (or PA01224b); Track 1: Consensus sequence; Tracks 2–10: Sequence variations of the two variants of PA01224a and/or P101224b compared with PA15511, PA05682, S1133, 1733, 138, AVS-B, MN9 and J18, respectively; Track 11: Sequencing depth of NGS, the axis of the coverage track corresponds to 0, 100, 200, 300, 400, and 500 reads from inside to outside; Track 12: Assembled contigs using de novo assembly (SPAdes); Track 13: Open reading frames (ORFs); Track 14: σC gene Sanger sequencing results; Track 15: Sequence variations between NGS and Sanger sequencing results in the σC gene.
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f2: Circos plot descriptions of the complete genomes of the two co-infection variant strains.(A) Reo/PA/Layer/01224a/14 variant (or PA01224a); (B) Reo/PA/Layer/01224b/14 variant (or PA01224b); Track 1: Consensus sequence; Tracks 2–10: Sequence variations of the two variants of PA01224a and/or P101224b compared with PA15511, PA05682, S1133, 1733, 138, AVS-B, MN9 and J18, respectively; Track 11: Sequencing depth of NGS, the axis of the coverage track corresponds to 0, 100, 200, 300, 400, and 500 reads from inside to outside; Track 12: Assembled contigs using de novo assembly (SPAdes); Track 13: Open reading frames (ORFs); Track 14: σC gene Sanger sequencing results; Track 15: Sequence variations between NGS and Sanger sequencing results in the σC gene.

Mentions: After de novo assembly using the SPAdes program, the clean reads generated a total of 52 contigs, varying from 109 to 3942 nt in length. After BLASTN searching of the 52 contigs, 19 contigs (Table 1) were identified to be ARV sequences, and 9 of the 10 ARV genome segments were targeted by two homologous contigs, except M2 targeted by one contig. These findings indicated there were two genomes of ARV variants with 9 different genome segments and one same M2 segment in the sequencing sample (Table 1). The sequencing depth at every base of the contigs was shown on track 11 of the circos plots (Fig. 2A,B). Highest similarity searching of the 19 ARV-related contigs in GenBank revealed that all 19 contigs had different homologies with other published reference ARV strains (82–98%). To obtain the sequencing coverage data, the sequencing reads were mapped back to the assembled contigs. The reads coverage was calculated from 26.82× to 254.01× on average for each segment. The mapped reads of each segment varied from 404 to 5978 reads, which positively correlated with the sequencing coverage data (Table 1). To identify the intra-host single-nucleotide variants (iSNVs) in the assembled contigs, the reads mapping results were processed using the resequencing program of CLC Genomics Workbench software with a 0.4% sequencing error correction. A total of 21 iSNVs were determined in five contigs corresponding to L1, L3, M1, S2, S3 and S4 segments (Table 1), and 17 of the 21 iSNVs had sequencing depths greater than 100×.


Detection and characterization of two co-infection variant strains of avian orthoreovirus (ARV) in young layer chickens using next-generation sequencing (NGS).

Tang Y, Lin L, Sebastian A, Lu H - Sci Rep (2016)

Circos plot descriptions of the complete genomes of the two co-infection variant strains.(A) Reo/PA/Layer/01224a/14 variant (or PA01224a); (B) Reo/PA/Layer/01224b/14 variant (or PA01224b); Track 1: Consensus sequence; Tracks 2–10: Sequence variations of the two variants of PA01224a and/or P101224b compared with PA15511, PA05682, S1133, 1733, 138, AVS-B, MN9 and J18, respectively; Track 11: Sequencing depth of NGS, the axis of the coverage track corresponds to 0, 100, 200, 300, 400, and 500 reads from inside to outside; Track 12: Assembled contigs using de novo assembly (SPAdes); Track 13: Open reading frames (ORFs); Track 14: σC gene Sanger sequencing results; Track 15: Sequence variations between NGS and Sanger sequencing results in the σC gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835796&req=5

f2: Circos plot descriptions of the complete genomes of the two co-infection variant strains.(A) Reo/PA/Layer/01224a/14 variant (or PA01224a); (B) Reo/PA/Layer/01224b/14 variant (or PA01224b); Track 1: Consensus sequence; Tracks 2–10: Sequence variations of the two variants of PA01224a and/or P101224b compared with PA15511, PA05682, S1133, 1733, 138, AVS-B, MN9 and J18, respectively; Track 11: Sequencing depth of NGS, the axis of the coverage track corresponds to 0, 100, 200, 300, 400, and 500 reads from inside to outside; Track 12: Assembled contigs using de novo assembly (SPAdes); Track 13: Open reading frames (ORFs); Track 14: σC gene Sanger sequencing results; Track 15: Sequence variations between NGS and Sanger sequencing results in the σC gene.
Mentions: After de novo assembly using the SPAdes program, the clean reads generated a total of 52 contigs, varying from 109 to 3942 nt in length. After BLASTN searching of the 52 contigs, 19 contigs (Table 1) were identified to be ARV sequences, and 9 of the 10 ARV genome segments were targeted by two homologous contigs, except M2 targeted by one contig. These findings indicated there were two genomes of ARV variants with 9 different genome segments and one same M2 segment in the sequencing sample (Table 1). The sequencing depth at every base of the contigs was shown on track 11 of the circos plots (Fig. 2A,B). Highest similarity searching of the 19 ARV-related contigs in GenBank revealed that all 19 contigs had different homologies with other published reference ARV strains (82–98%). To obtain the sequencing coverage data, the sequencing reads were mapped back to the assembled contigs. The reads coverage was calculated from 26.82× to 254.01× on average for each segment. The mapped reads of each segment varied from 404 to 5978 reads, which positively correlated with the sequencing coverage data (Table 1). To identify the intra-host single-nucleotide variants (iSNVs) in the assembled contigs, the reads mapping results were processed using the resequencing program of CLC Genomics Workbench software with a 0.4% sequencing error correction. A total of 21 iSNVs were determined in five contigs corresponding to L1, L3, M1, S2, S3 and S4 segments (Table 1), and 17 of the 21 iSNVs had sequencing depths greater than 100×.

Bottom Line: The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins.Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains.Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains.

View Article: PubMed Central - PubMed

Affiliation: Wiley Lab/Avian Virology, Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, United States.

ABSTRACT
Using next-generation sequencing (NGS) for full genomic characterization studies of the newly emerging avian orthoreovirus (ARV) field strains isolated in Pennsylvania poultry, we identified two co-infection ARV variant strains from one ARV isolate obtained from ARV-affected young layer chickens. The de novo assembly of the ARV reads generated 19 contigs of two different ARV variant strains according to 10 genome segments of each ARV strain. The two variants had the same M2 segment. The complete genomes of each of the two variant strains were 23,493 bp in length, and 10 dsRNA segments ranged from 1192 bp (S4) to 3958 bp (L1), encoding 12 viral proteins. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of all 10 genome segments revealed 58.1-100% and 51.4-100% aa identity between the two variant strains, and 54.3-89.4% and 49.5-98.1% aa identity between the two variants and classic vaccine strains. Phylogenetic analysis revealed a moderate to significant nt sequence divergence between the two variant and ARV reference strains. These findings have demonstrated the first naturally occurring co-infection of two ARV variants in commercial young layer chickens, providing scientific evidence that multiple ARV strains can be simultaneously present in one host species of chickens.

No MeSH data available.


Related in: MedlinePlus