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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Infect Genet Evol. 2015 Apr 1;32:313–321. doi: 10.1016/j.meegid.2015.03.029

Genomic characterization of a turkey reovirus field strain by Next-Generation Sequencing

Yi Tang 1, Huaguang Lu 1,*, Aswathy Sebastian 2, Yin-Ting Yeh 3, Craig A Praul 4, Istvan U Albert 2, Si-Yang Zheng 3
PMCID: PMC4649943  NIHMSID: NIHMS733189  PMID: 25841748

Abstract

The genome of a turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13), isolated from a turkey flock in Pennsylvania (PA) in 2013, has been sequenced using Next-Generation Sequencing (NGS) on the Illumina MiSeq platform. The genome of the PA TARV field strain was 23,496bp in length with 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments ranged from 1,192bp (S4) to 3,959bp (L1). The 5’ and 3’ conserved terminal sequences of the PA TARV field strain were similar to the two Minnesota (MN) TARVs (MN9 and MN10) published recently and avian orthoreovirus (ARV) reference strains. Phylogenetic analysis of the nucleotide sequences of all 10 genome segments revealed that there was a low to significant nucleotide sequence divergence between the PA TARV field strain and reference TARV and ARV strains. Analysis of the PA TARV sequence indicates that this PA TARV field strain is a unique strain and is different from the TARV MN9 or MN10 in M2 segment genes and ARV S1133 vaccine strain.

Keywords: Genome segments, genotyping, Next-Generation Sequencing, turkey arthritis reovirus

1. Introduction

Avian orthoreoviruses (ARV) belong to the genus Orthoreovirus in the family Reoviridae. They are non-enveloped viruses and contain a double-stranded RNA (dsRNA) genome with 10 segments. The viral genome is enclosed within a double protein capsid shell with a diameter of 70-80nm (Spandidos and Graham, 1976; van der Heide, 2000). Polyacrylamide gel electrophoresis indicates that the whole genome can be separated into 10 genomic segments including 3 large (L1, L2, L3), 3 medium (M1, M2and M3) and 4 small (S1, S2, S3 and S4) segments (Nick et al., 1975; Varela and Benavente, 1994). There are 8 structural proteins (λA, λB, λC, μA, μB, σA, σB and σC) and 4 non-structural proteins (μNS, P10, P17 and σNS) encoded by the segmented genome (Varela et al., 1996).

ARV is one of the most important avian viruses causing clinical diseases in poultry worldwide. ARV affected poultry flocks commonly suffer from viral arthritis/tenosynovitis, enteric disease, immunosuppression, runting-stunting syndrome, and malabsorption syndrome (Sterner et al., 1989; van der Heide, 2000). Most domestic avian species are susceptible to ARV infections, such as broiler breeders (Ide and Dewitt, 1979), layer breeders (De Gussem et al., 2010), broilers (Howell and Walker, 1972), goose (Bezerra et al., 2012; Palya et al., 2003), turkey (Giangaspero et al., 1997; Taber et al., 1976), duck (Baroni et al., 1980; Petek et al., 1973; Rey et al., 1999), pigeon (Vindevogel et al., 1982), and quail(Guy et al., 1987; Magee et al., 1993; Ritter et al., 1986). However, the meat-type chickens are well documented species that are more susceptible to the viral arthritis/tenosynovitis caused by ARV than other avian species (De Gussem et al., 2010).

The outbreaks of turkey arthritis reovirus (TARV) infections initially occurred in the United States in late 1970's and 1980's (Afaleq and Jones, 1989; Levisohn et al., 1980). The recent TARV outbreaks started in Minnesota (MN) in 2009. Research findings by Mor's group suggested that the MN TARV strains obtained from these outbreaks were likely the tropism of ARV variants re-involved with arthritis in turkeys (Mor et al., 2013). A similar TARV infection problem occurred in a commercial turkey flock at 9 weeks of age in Pennsylvania (PA) in summer 2011, and another flock (17 weeks old) of the same company was infected a week later. The affected birds suffered from severe lameness and tenosynovitis, tendon rupture or hemorrhage. Tendon and synovial tissues collected from sick birds were virus isolation positive for TARV. The 2011 TARV cases diagnosed in PA were the first index cases representing TARV infections in the east region of United States, and similar TARV cases were continuously diagnosed in 2013, 2014 and 2015.

Until recently, research findings in TARV sequencing studies, mostly based on the L-class and S-class genomic segments, showed a great genetic divergence between TARVs and ARV vaccine strains (Mor et al., 2014a; Mor et al., 2014b). Other research studies indicated that highly pathogenic ARV variants were capable of infecting not only unvaccinated broiler chickens, but also vaccinated broiler breeders (Dandar et al., 2013; Rosenberger et al., 2013; Troxler et al., 2013). In this paper, we report the complete genome sequence of a PA field strain (Reo/PA/Turkey/22342/13), isolated from TARV infected turkeys showing severe arthritis and lameness in a commercial turkey flock at 14 weeks of age, by using Next-Generation Sequencing (NGS) with the Illumina MiSeq.

2. Materials and methods

2.1 The TARV strain and RNA sample preparation

The PA TARV field strain (Reo/PA/Turkey/22342/13) used in this study was isolated from tendons and synovial tissues of sick birds showing arthritis/tenosynovitis in a PA commercial turkey flock at 14 weeks of age in 2013. Virus isolation and propagation were conducted in LMH (Leghorn Male-chicken Hepatocellular-carcinoma, ATCC CRL-2113) cell cultures as routine cell culture procedures (Heggen-Peay et al., 2002). Viral RNA extraction from the TARV infected cell culture fluid was carried out with a total RNA extraction Kit (QIAGEN, Valencia, CA, USA) following manufacturer's instructions.

2.2 Sequencing

Libraries were constructed from total RNA samples using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according the manufacturer's protocol with the exception that the initial poly A enrichment step was skipped. Library size and quality was assessed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Library concentration was assessed by qPCR using the KAPA Library Quantification Kit Illumina Platforms (Kapa Biosystems, Wilmington, MA, USA. Libraries were sequenced on an Illumina MiSeq using 150 nt single read sequencing according to the manufacturer's protocol (Illumina, San Diego, CA, USA).

2.3 Viral genome assembly

The overall pipeline for sequence data analysis started with raw NGS reads (Fig. 1). Briefly, the first step was to trim off the adaptors added for sequencing and remove reads mapping to contaminants (e.g., mRNA, rRNA, chicken and human sequences) using both sortMeRNA (Kopylova et al., 2012) and BWA-MEM (Li and Durbin, 2010) methods. Unmatched sequence reads were assembled using de novo SPAdes assembly software (ver 3.5.0) (Bankevich et al., 2012). All the assembled contiguous sequences (contigs) were aligned to the reference genome using LASTZ (Harris, 2007) to identify and extract maximally aligned viral contigs. To further improve the contigs, all raw reads of each segment were mapped back to the assembled contigs. Finally, the consensus sequences from the re-mapping reads and LASTZ contig alignment were obtained using SAMtools commands (Li et al., 2009).

Fig. 1.

Fig. 1

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A flow chat of genome sequencing steps for the turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13) detected in Pennsylvania (PA) of the USA. The left is data analysis pipeline for viral genome assembly; The right is a pie chart of the homology search results for NGS reads.

2.4 Obtaining 5’ and 3’ termini

The rapid amplification cDNA ends (RACE) methods were used to obtain the 5’ and 3’ termini for each of the 10 genome segments. A short oligonucleotide PC3, which was phosphorylated at the 5’ end and blocked at the 3’ end with dideoxy cytosine, was ligated to the 3’ ends of extracted the genomic RNA (Watson et al., 1992). The ligation reaction was performed by T4 RNA ligase (New England Bio Labs, Ipswich, MA, USA). Following the incubation, the ligated dsRNA was purified using agarose gel extraction columns following the manufacturer's instructions (Lot No. 04113KE1, Axygen, Tewksbury, MA, USA). Subsequently, the PC2 complementary primer to the ligated oligonucleotide was combined with gene specific primers in different reactions for 5’ and 3’ ends of each genomic segment amplification and sequencing, respectively, using the conditions as described above. The DNA concentration of the purified PCR product was measured using a NanoDrop™1000 (Thermo Scientific, Waltham, MA, USA) spectrophotometer and then submitted to Penn State Genomics Core Facility for Sanger sequencing.

2.5 Sequence analyses

Lasergene 12 Core Suite (DNASTAR, Inc. Madison, WI, USA) was used for Sanger sequencing results assembly, viral ORFs prediction and nucleotide (nt) sequences translation. Sequence similarity was evaluated using BLASTN search in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignments of sequences were carried out using the ClustalW 1.83 program (http://align.genome.jp/). Neighbor-joining and maximum-likelihood (ML) phylogenetic trees were generated and tree topologies were validated by bootstrap analysis as implemented in MEGA program (Version 5.0) with absolute distances following 1,000 bootstrap replicates (Tamura et al., 2011). Visualizing of NGS and viral genomic data plot were generated by the Circos method (Krzywinski et al., 2009). The S1 gene ORF organization mapping was performed using CLC Genomic Workbench V7.5 software (QIAGEN, Boston, MA, USA). Analysis of whole genome alignments was performed using the mVISTA online platform (http://genome.lbl.gov/vista/mvista/submit.shtml). In order to conduct genome comparison of this PA TARV field strain with other reference strains, full genomic sequences of two MN turkey TARV strains (MN9, MN10) and 6 ARV reference strains (S1133, 1733, 138, 176, AVS-B and J18) retrieved from GenBank (Table S1) were used for comparison analysis.

3. Results

3.1 Viral RNA extraction and RT-PCR confirmation of the PA TARV field strain

The PA TARV field strain was freshly propagated in LMH cell cultures for viral RNA extraction in this study, and the extracted RNA was confirmed positive by the S1-based RT-PCR using P1/P4 primers to amplify 1088bp of the S1 gene sequence (Kant et al., 2003).

3.2 Summary of NGS data of PA TARV field strain genome

From the total RNA sample of the PA TRAV filed strain, a total of 1,686,331 reads of length 35-151 nt- following trimming were obtained resulting in 524 Mb of fastq format sequence data. Sequence reads that passed default quality control (QC) filters on the MiSeq platform were aligned to reference sequences of chicken genomic DNA and rRNA databases followed by quality trimming to remove contamination reads and excluding reads with similarities to chicken mRNA or rRNA sequences. As a result, 310,284 reads (18.4%) were identified to be the chicken mRNA source, 1,305,052 reads (77.4%) to be the chicken rRNA source, and 8432 reads (0.5%) to be sequencing adapter (Fig. 1). The remaining 63,935 reads (3.7%) were was considered as clean reads and further analyzed using a BLASTN procedure, which revealed 47,217 reads (2.8%) as no hits and 16,718 reads (0.9%) as orthoreovirus (Fig. 1).

3.3 De novo assembly of the PA TARV field strain genome

All clean reads from the QC step, including no hits reads, were assembled using SPAdes de novo assembler. After BLASTN searching, a total of 10 contigs were generated by software according to the 10 ARV genome segments. Among all the 10 contigs, the whole lengths of each segment were correctly assembled as the single contig (Table 1 and Fig. 2). Similarity comparison in nt sequences of the 10 contigs to published ARV reference strains from Genbank indicated that all 10 contigs had different homologies with other published reference ARV strains. The highest similarities (sharing 95%-98% nt identities) for general contigs identification were seen between the PA TARV field strain and various classic reference strains (Table 1). To obtain the sequencing depth data and identify single nucleotide variations (SNVs) in the 10 segments, the sequencing reads were mapped back to the assembled contigs. The coverage at every base of the segments is shown on the track 6 of the circos plot (Fig. 2). Read coverage was calculated at 117× on average for all segments. To identify SNVs in each genomic segment of the PA TARV field strain, alignment results were screened using CLC Genomic Workbench software. As a result, a total of 13 SNVs were identified in five genomic segments in the L or M class, and 0 in the S class. Each of the 13 SNVs had a read depth of 100× or above (Table 1).

Table 1.

De novo assembly contigs of the Pennsylvania turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13)

Segment GenBank Accession No. Contig length (bp) Highest similarity to TARV reference strain in GenBank Identities (%) SNVs Reads Contig location
L1 3882 Turkey/USA/MN/2011/TERV-MN6
L1 λA gene (KJ865912)		 96 3 2919 22-3903
L2 3805 Turkey/USA/MN/2011/TARV-MN2
L2 λB gene (KJ865893)		 92 2 3118 14-3819
L3 2883 Turkey/USA/MI/2013/TARV-MN12
L3 λC gene (KJ865888)		 98 1 2755 16-3898
Ml 2199 Turkey/USA/MN/2014/TARV-MN13
μA gene (KJ874318)		 95 5 1897 13-2211
M2 2158 Turkey/USA/MI/2013/TARV-MN12
μB gene (KJ874294)		 99 0 882 12-2150
M3 1909 Turkey/USA/MN/2011/TERV-MN3
μNS gene (KJ874275)		 95 2 1943 25-1932
S1 1597 TARV MN3
S1 p10, p17, σC genes (KF872234)		 99 0 1003 15-1611
S2 1308 TARV MN10
σA gene, partial (KF872254)		 95 0 466 10-1317
S3 1172 TK/MN/USA/D-AB15/2011
S3 σB gene (KF183931)		 97 0 1075 18-1189
S4 1169 turkey/MO/SEP-828/05
S4 σNS gene (EU400282.1)		 97 0 662 13-1181
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Fig. 2.

Fig. 2

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Circular representation of full genome of the PA TARV field strain (Reo/PA/Turkey/22342/13). Track 1: Consensus sequence; Track 2: Open reading frames (ORFs); Track 3: σC gene sanger sequencing result; Track 4: Sequence variations between NGS and Sanger sequencing result in σC gene; Track 5: Assembled contigs by de novo assembly (SPAdes); Track 6: Sequencing depth of NGS, the axis of coverage track corresponds to 0,110,220,330,440,550 reads from inside to outside; Track 7-11: Sequence variations of the PA TARV (Reo/PA/Turkey/22342/13) in comparison with 2 MN TRAVs of MN9, MN10, and 3 ARV reference strains of 138, S1133 and AVS-B, respectively.

3.4 The complete genome of the PA TARV field strain

To determine the 5’ and 3’ termini of each segment not included in NGS assembly contigs, we used the RACE technique combined with Sanger sequencing, thus we successfully obtained the full length sequence of the PA TRAV field strain. The complete sequences of all 10 segments of the PA TARV field strain were finally obtained when the 5’ and 3’ termini Sanger sequencing results from each of the 10 segments were all combined. The full-length sequences of all 10 segments of the PA TARV field strain were deposited into GeneBank in November 2014 (Genbank accession numbers: KP173683 to KP173692, Reo/PA/Turkey/22342/13, Table 1). The genome of the PA TARV field strain is 23,496bp in length with an approximately 50% G+C content and 10 dsRNA segments encoding 12 viral proteins. The lengths of the genomic segments range from 1192bp (S4) to 3959bp (L1) and are similar to those TAVR and ARV reference strains. ORF analysis of nucleotide sequences indicated that 9 of the 10 genome segments (except S1) encoded one single ORF. The 1645bp S1 segment was shown to be tricistronic with partially overlapping ORFs encoded p10 at 25–324bp, p17at 296-748bp and σC at 633-1613bp, respectively. The length of untranslated regions (UTRs) ranges from 12-30bp at the 5’ ends, and from 33-98bp at the 3’ ends (Table 2). Other segments coding sequences were predicted to encode 9 putative proteins (λA on L1, λB on L2, λC on L3, μA on M1, μB on M2, μNS on M3, σA on S2, σB on S2, and σNS on S3) with sizes ranging from 368 amino acid (aa) to 1294 aa. All of the 10 fragments share a GCUUUU motif in their 5’ UTR and a UCAUC motif in their 3’ UTR. The 5’ UTRs of the PA TARV field strain are the same as the corresponding sequences in ARV reference strains, but different from other species within the genus (Table 3). The last 5 bases of 3’ UTRs sequences of all orthoreoviruses were found to be highly conserved with a UCAUC motif. Moreover, the first and last nucleotides of each segment in TARV were complementary (G-C).

Table 2.

General genome features of the Pennsylvania turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13), all 10 genome segments and GenBank accession numbers

Genome segment Size Length (nt) of the Sequence at the termini Protein size(aa) Encoded protein
5’ end ORF 3’ end 5’ end 3’ end
L1 (KP173683) 3959 21 3882 56 GCUUUU UCAUC 1294 λA(core shell)
L2 (KP173684) 3907 12 3858 37 GCUUUU UCAUC 1286 λB(core RdRp)
L3 (KP173685) 3829 14 3780 36 GCUUUU UCAUC 1260 λC(core turret)
Ml (KP173686) 2283 12 2199 72 GCUUUU UCAUC 733 μA(core NTPase)
M2 (KP173687) 2158 29 2031 98 GCUUUU UCAUC 677 μB(outer shell)
M3 (KP173688) 1996 24 1908 64 GCUUUU UCAUC 636 μNS(NS factory)
S1 (KP173689) 1645 24 300 33 GCUUUU UCAUC 100 p10(NS FAST)
453 151 p17(NS other)
981 327 σC(outer fiber)
S2 (KP173690) 1324 15 1251 58 GCUUUU UCAUC 417 σA(core clamp)
S3 (KP173691) 1203 30 1104 68 GCUUUU UCAUC 368 σB(outer clamp)
S4 (KP173692) 1192 23 1104 65 GCUUUU UCAUC 368 σNS(NS RNAb)
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Table 3.

Comparison of segment 5’ and 3’ non-coding regions of the Pennsylvania turkey arthritis reovirus (TARV) field strain (Reo/PA/Turkey/22342/13) among members of Orthoreovirus genus

Orthoreovirus species Host Terminal region sequences (5’ to 3’)
Reo/PA/Turkey/22342/13 Turkey GCUUUUU...UAUUCAUC
Avian orthoreovirus (ARV)-S1133 Broiler Chicken GCUUUUU...UAUUCAUC
Avian orthoreovirus (ARV)- J18 Muscovy Duck GCUUUUU...UAU/CUCAUC
Broome virus (BroV) Little Red Flying Fox GUCAA...UCAUC
Baboon orthoreovirus (BRV) Yellow Baboon GUAAA...UCAUC
Nelson bay virus (NBV) Grey-headed Flying Fox GCUUUA...UCAUC
Pulau virus (PuV) Fruit Bat GCUUUA...UCAUC
Melaka virus (MELV) Human GCUUUA...UCAUC
Mammalian orthoreovirus (MRV)-1 Mink GCUA...UCAUC
Mammalian orthoreovirus (MRV)-2 Human GCUA...UCAUC
Mammalian orthoreovirus (MRV)-3 Masked Civet Cats GCUA...UCAUC
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Genomic analysis of the PA TARV field strain revealed that the lengths of all 10 genomic segments were almost identical to the cognate segments of previously reported TARVs and standard ARV reference strains. Each genome segment started and ended with short and highly conserved sequences, however, of which the sequence at the 5’ end (GCUUUU) was different from the sequences determined for other orthoreovirus species. Whereas, the sequence at the 3’ end (UCAUC) was a typical segment termination motif and highly conserved within Orthoreovirus genus, thus this PA TARV field strain genome segments belonged to a particular viral species with no segment reassortment between ARV and mammalian reoviruses (MRV). Furthermore, similarity searches using nt and aa sequences of the PA TARV field strain indicated that all 10 segments were highly conserved in the homologous proteins of reference strains, and some of the genomic features were also found in the J18 duck strain. All these sequence data indicate that the PA TARV field strain belongs to the ARV species.

3.5 Sequence comparison

The PA TARV field strain nt and aa sequences were compared to their homolog with two TARVs (MN9 and MN10) and three ARV reference strains (S1133, 138 and AVS-B). The nt sequence variations are visible on the track 7, 8, 9, 10, 11 of the circos plot (Fig. 2); and different identity values (Table S2) are seen between these strains and their genome segments. The PA TARV strain was found to be different from the MN9 and MN10 strains at the M2 segment μB encoding gene because they shared only 75.5% nt identities and 89.7% aa identities; while they shared high sequence identities at the other 9 segments (nt: 91.4–98.7%; aa: 97.1-99.0%) (Table S2). When the PA TARV field strain was compared with the 3 ARV strains (S1133, 138, AVS-B): their aa and nt sequence identities were remarkably low at S1 segment σC encoding genes (48.3%-59.3%), low at S3 segment σB encoding genes (70.1%-79.2%) and S4 segment σNS encoding genes (74.2%-77.8%); and moderate to high at all other segments (>80%) except L3 segment (nt: 72.5%) of the S1133 strain (Table S2). The σC ORF and the encoded proteins of the one PA and two MN TARV field strains were seen to be highly conserved in length in comparison with ARV strains (Fig. 3), but its length was 5aa longer than the J18 ARV. The predicted sizes of p10 and p17 ORFs of the PA TARV field strain are 99aa and 150aa, 1-3aa and 4-10aa, respectively, which are longer than the corresponding ORF regions of ARV strains.

Fig 3.

Fig 3

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Comparisons of ORF organizations of S1 segments between the PA TARV field strain (Reo/PA/Turkey/22342/13) and 5 ARV reference strains. Yellow color arrow = p10 ORF, blue color arrow = p17 ORF, pink color arrow = σC ORF. The beginning and ending nucleotide numbers were labeled on each arrow. (Note: S1 sequence comparison was not done with other TARVs due to the lack of complete S1 segment sequences)

3.6 Phylogenetic analyses

The evolutionary relationships of the PA TARV field strain with the Orthoreovirus genus members, particularly other ARV strains and TARV strains, were determined by phylogenetic analysis. The phylogenetic trees (Fig. 4) were constructed based on nt sequences of 3 L and 3 M genome segments and 4 σ-class genes using the maximum likelihood method with bootstrapping. All the phylogenetic trees illustrated a great divergence between ARV, TARV and MAR strains. For most genomic segments or genes, the PA TARV field strain and the MN TARV strains formed a turkey-origin TARV group that was different from the chicken-origin ARV strains and duck-origin ARV strains. The TARV group showed a closer evolutionary relationship with chicken ARV strains than duck ARV in all segments or genes except M2. In the M2 phylogenetic tree, four TRAV strains including PA TARV field strain divided into group 1 and group 2. The PA TARV field strain and MN12 form TARV group 1, which is closely related to chicken and duck ARV strains but is distinct from the TARV group 2 (formed by MN9 and MN10) with more than 14% nt divergence. Phylogenetic analyses also revealed that the segments or genes encoding for outer capsid proteins (μB, σB and σC) exhibitnoticeably higher divergence than other segments for ARVs as indicated in their sequence comparisons.

Fig. 4.

Fig. 4

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Phylogenetic trees constructed by avian reopvurus (ARV) and mammalian reovirus (MRV) based on nucleotide sequences of the 10 (3L, 3M and 4 S) homologous genome segments of orthoreovirus species. Blue color= chicken-origin ARV, red color= turkey-origin ARV or TARV, green color=duck-origin ARV, and purple color=MRV; the PA TARV field strain (Reo/PA/Turkey/22342/13) marked in red dot.

3.7 Overall genomic constellation of the TARV

Figure 5 illustrates the sequence similarity values and divergence of individual genes and their encoded proteins in comparison of the PA TARV field stain with 2 TARV and 6 ARV reference strains. The greatest sequence similarity values were seen in 9 of the 10 genome segments of TARV MN9 or MN10 strain, with the exception of M2 segment which showed high diversity. However, the highest identity (>90% nt) of M2 segment was found between the PA TARV field stain and chicken ARV-1733 strain. Considerable genetic relatedness of the PA TARV field strain and ARV reference strains was found along 9 of the 10 genome segments, with exception for the 3’ end of the S1 segment corresponding to the σC coding region. The concatenated genes revealed that only L1, L2, M1, M3 and S2 genome segments were found to be closely related between PA TARV field strain and the chicken-origin ARV reference strains, while the remaining 5 genome segments appeared to be more divergent, particularly for the L3, S1 and S3 segments. In addition, L3, S1, S3 and S4 segments of the PA TARV field strain and S1133 strain were only low to moderately related to each other, which indicated that the PA TARV field strain was very diverse from the ARV vaccine strain. In comparison with the duck-origin ARV J18, the PA TARV field strain shared very low sequence identities with it through the whole genome, particularly the S1 segments (<50%).

Fig. 5.

Fig. 5

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The mVISTA method for whole genome nucleotide alignment; this figure illustrate alignment results of the PA TARV field strain (Reo/PA/Turkey/22342/13) in comparisons with 2 MN TARVs (MN9, MN10) and 6 ARV reference strains (138, S1133, AVS-B, 1733,176 and J18) retrieved from GenBank (Table S1); Areas in pink color represent ≥ 90% similarities; and areas in white represent < 90% similarities. The scale bar measures approximate length of the concatenated genome.

4. Discussion

ARV was first identified as the causative agent to a highly pathogenic poultry disease characterized as arthritis and tenosynovitis (Olson and Kerr, 1966; Olson et al., 1957; Walker et al., 1972). Thereafter, many ARV isolations from various locations in the United States were reported, and ARV infection in poultry were well described and documented (Deshmukh and Pomeroy, 1969; Johnson, 1972; Van der Heide et al., 1974). Extensive ARV infections in PA poultry have been continuously diagnosed in our laboratory from 2011 to the present time. We have made over 300 ARV isolates so far, mostly from broilers and turkeys, some from layers and a small number from other avian species. We will publish our research findings on these ARV isolates soon. Nonetheless, a complete genomic characterization of a PA broiler ARV field strain, by using traditional genome sequencing procedures (Banyai et al., 2011), was published recently (Tang and Lu, 2015).

Until recently, research findings in TARV sequencing studies, mostly based on the L-class and S-class genomic segments, showed a great genetic divergence between TARVs and ARV vaccine strains (Mor et al., 2014a; Mor et al., 2014b). Other research studies indicated that highly pathogenic ARV variants were capable of infecting not only unvaccinated broiler chickens, but also vaccinated broiler breeders (Dandar et al., 2013; Rosenberger et al., 2013; Troxler et al., 2013). In this study, we employed Illumina NGS technology to characterize the complete genome of the PA TARV field strain. By de novo assembling the viral genome, the10 contigs corresponding to the 10 genomic segments of the PA TAVR field strain were generated. The assembled contigs covered the most areas of the 10 genomic segments, only missed the genomic termini. In NGS, the genomic termini are often under-represented in the obtained viral reads, which is likely due to a variety of reasons including the RNA second structure and protein binding site at genomic termini, viral RNA population, reads trimming before assembly, and de novo assembly software error. Specifically designed primers in genomic termini for cDNA synthesis have improved the number of termini sequences reads, but have not completely resolved the problem (Marston et al., 2013). When mapping reads back to the consensus sequence, the coverage was observed at 117× on average for all segments, indicating a sufficient redundancy to identify the SNVs in viral sequences (Kuroda et al., 2010; Kwok et al., 2012). In this study, a total of 13 SNV sites of the PA TARV field strain were observed in consensus sequence presenting in the low level population. This approach is also useful to determine high proportion SNV changes within the same virus strain using consensus sequencing, which can be targeted directly by deep sequencing the relevant genomic regions (Marston et al., 2013).

Sequence comparison of all S-class encoding genes between the PA TARV field strain and MN9 or MN10 TARVs revealed that they shared very high nt (>93.4%) and aa (>97.2%) sequence identities . Whereas, when compared with ARV reference strains of S-class encoding genes, the PA TARV field strain shared very low sequence identities with them in σC gene (nt:<54.2%; aa< 59.3%) and low in σB and σNS genes (nt: <70.1%; aa: <78.4%). In σA gene, the shared sequence identities of the PA TARV field strain with the 138 strain (nt: 92%; aa: 97.6%) were higher than with other ARV strains (nt: 88.9%; aa: 97.3%), which indicated that ARV 138 strain was more closely related to the PA TARV field strain than other ARVs. The sequence divergences of the σB and σC encoding genes were significantly higher than those of other genes between TARVs and ARVs, which should be reasonable true because the σB and σC proteins are components of the ARV virion outer capsid (Benavente and Martinez-Costas, 2007). Particularly, σC gene displayed the feature of highest level of sequence divergence and rapid evolution; therefore, the σC gene could be used as a genetic marker for rapid differentiation between ARV and TARV.

The highest identity rates were found in L2 segment coding λB protein between the PA TARV field strain and ARV reference strains, which is likely because the λB is the highly conserved viral RNA-dependent RNA polymerase (RdRp) protein (Xu and Coombs, 2008). Sequence comparisons among the M-class genes demonstrated that the sequence divergences of the M2 segment and the μB protein were higher than those of the other M-class genes and their encoded proteins. Sequence identities shared between the PA TARV field strain and MN TARVs were low in μB encoding gene (nt: 75.5%; aa: 89.7%) which is not unexpected because the position of the μB, like σB and σC, in the outer capsid probably results in more immune selection on the μB than μA and μNS. Selection pressures might restrict variability within the μB, σB and σC genes to be included in the requirement to form a stable interaction with each other. M2 sequences of additional 17 strains (14 turkey-origin and 3 chicken-origin) including MN6 (KJ874301) and MN12 (KJ874294) retrieved from GeneBank were used for nt similarity comparison. Our blast results showed that they all shared high similarities (nt>91%) at M2 segment with the PA TARV field stain in this study. Sequence comparisons of other segments with these reference strains may reveal differences between them; especially full genome sequence comparisons between TARVs should be conducted. However, such comparisons are not available currently due to the lack of all 10 segments or full genome sequences of other TARVs.

Phylogenetic analysis of individual segments and genes revealed various patterns of clustering with reference strains, and such relationship was supported by high bootstrap values. In general, the nt and aa sequence identities should be greater than 75% and 85%, respectively, between homologous orthoreovirus genes or within a same species group; whereas, if the nt and aa identities are less than 60% and 65%, respectively, between distinct species groups (Urbano and Urbano, 1994). For most genomic segments, the PA TARV field strain clustered together with MN TARV strains. However, in M2 segment phylogenetic analysis, 4 TARVs were evolved into two genetically heterogenetic groups, revealing the existence of at least two predominant TARV genotypes. The phylogeny of sigma-class genes showed that the TARV strains formed a different cluster from ARV reference strains, whereas the TARV strains were only distantly related to the 091, J18 and D20-99 waterfowl strains. In σC gene, this PA TARV and 3 MN TARVs formed a separated cluster, which was different from most chicken-origin and duck-origin ARVs. Although the σC gene phylogenetic relationships between TARVs and the ARV GEL13a98M strain (Kant et al., 2003) were close, the host species were different, the TARV was a turkey-origin, the GEL13a98M strain was a chicken origin, cross infections each other of the two species remained unclear. These findings suggested that reassortment between the ARVs from malabsorption syndrome and ARVs from viral arthritis could have played a role in the origin of the TARVs emerged previously in the USA and re-emerging recently, which agreed with a number of published research studies on ARV genes (Day et al., 2007; Hsu et al., 2005; Su et al., 2006). Collectively, all 10 segment-based phylogenies permitted insight into the origin of the unique genetic configuration of the PA TARV field strain in this study. This PA TARV field strain implies a greater genetic diversity than documented ARV reference strains, thus it is a novel or newly emerging TARV strain, similar as MN9 or MN10 TARVs.

Our research findings revealed that the complete genome of the PA TARV field strain exhibited distinct molecular features when compared with chicken-origin ARV and duck-origin ARV strains, and its M2 segment was remarkably different from the TARV MN9 or MN10 strains although they shared high similarities in other segments. These interesting results provide scientific insights into the evolutionary relationship between species origins and provide important genetic information to better understand stabilities or variations of TARV field strains. In conclusion, we determined the whole virus genome sequence and completed full genomic sequence characterization on this PA TARV field strain, which exhibits a number of different properties and pathogenicity compared with the members in Orthoreovirus genus. This is the first report that the TARV full genomic features were determined from a single virus strain.

Our research findings have demonstrated that this new PA TARV field strain shares high nt and aa sequences similarity and genomic features with MN9 or MN10 TARV strains. The topological heterogeneity observed among the phylogenetic trees and the sequence pairwise comparison results have suggested that genetic reassortment of the L, M and S segments might have occurred between re-emerging TARV strain and ARV strains. Therefore, full genomic analysis of the PA TARV field strain revealed that there were distinct molecular sequence characteristics in comparison with standard ARV vaccine strains. The research findings provide full genomic data for better understanding the evolutionary genomic changes of a newly emerging TARV field strains or variants.

Supplementary Material

Highlights.

  1. The Illumina Next-Generation Sequencing (NGS) was used to conduct full genome deep sequencing for a turkey arthritis reovirus (TARV) field strain.

  2. This is the first report that the TARV full genomic features were determined from a single virus strain.

  3. A total of 13 single nucleotide variation (SNV) sites were determined in the L and M genome segments of the TARV strain; 0 in the S segment.

  4. The predicted sizes of p10 and p17 ORFs of the TARV strain were longer than those of reference strains.

Acknowledgement

This research study was funded by the Pennsylvania Department of Agriculture, Animal Health and Diagnostic Commission's 2014 research project, PDA Contract ME No. 44134401; The National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, Grant No. UL1 TR000127.

Footnotes

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