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nomic characteristics of a novel reovirus from Muscovyckling in China

o Yun, Bin Yu, Zheng Ni, Weicheng Ye, Liu Chen, Jionggang Hua, Cun Zhang *

tute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

1. Introduction

Avian reoviruses (ARVs) are members of the Orthor-eovirus genus in the family Reoviridae. They are non-enveloped viruses that replicate in the cytoplasm ofinfected cells and contain a fragmented double-strandedRNA genome enclosed within a double protein capsid shellwith a diameter of 70–80 nm. The genomic segments canbe separated on the basis of their electrophoretic mobilityinto three size classes: large (L1–L3), medium (M1–M3),

T I C L E I N F O

le history:

ived 4 July 2013

ived in revised form 23 October 2013

pted 4 November 2013

ords:

el Muscovy duck reovirus

ence analysis

otype

CR assay

A B S T R A C T

A new reovirus was isolated from a sick Muscovy duckling with hemorrhagic-necrotic

lesions in the liver in Zhejiang, China in 2000 and was tentatively denoted a new type of

Muscovy duck reovirus (N-MDRV ZJ00M). This reovirus was propagated in a chicken

fibroblast cell line (DF-1) with obvious cytopathic effects. The reovirus’s genome was

23,419 bp in length with an approximately 50% G+C content and 10 dsRNA segments

encoding 12 proteins. The length of the genomic segments was similar to those of avian

reoviruses (ARVs), which range from 3959 nt (L1) to 1191 nt (S4) in size. All of the

segments have the conserved terminal sequences 50-GCUUUUU. . .UUCAUC-30 , and all of

the genome segments, with the exception of S1, apparently encoded one single primary

translation product. The genome analysis revealed that the S1 segment of N-MDRV is a

tricistronic gene that encodes the overlapping ORFs for p10, p18, and sC. This finding is

similar to that found for ARVs but distinct from that found for classical MDRV and GRV,

which have a bicistronic S4 segment that encodes p10 and sC and do not encode p18. The

amino acid (aa) alignments of the putative proteins encoded by the main ORF in each

segment revealed a high similarity (14.1–100%) to the counterpart proteins encoded by

other ARV species from the avian orthoreoviruses (e.g., ARV, classical MDRV and N-MDRV)

in the Orthoreovirus genus, particularly with N-MDRV (94.6–100%). The phylogenetic

analysis of the nucleotide sequences of all 10 genome segments revealed that N-MDRV

ZJ00M is distinct from all other described reovirus species groups but is a separated from

the ARV (including MDRV and GRV) species within orthoreovirus species group II and

grouped into the classical MDRV and GRV genogroup with the N-MDRV isolates. The

MDRV genogroup can be further divided into two genotype clusters. The morphological

and pathological analyses and the genetic characterization of N-MDRV ZJ00M suggest that

it belongs to genotype 2 (N-MDRV). In addition, the RT-PCR assays of DRV diseased

duckling and gosling samples collected from different regions of China during 2000–2013

indicate that N-MDRV is currently the prevalent genotype in China.

� 2013 Elsevier B.V. All rights reserved.

Corresponding author at: Institute of Animal Husbandry and

rinary Sciences, Zhejiang Academy of Agricultural Sciences, 145

iao Road, Hangzhou 310021, China. Tel.: +86 571 8640 4182;

+86 571 8640 0836.

E-mail addresses: zhangcun@aliyun.com, zhangcun@mail.zaas.ac.cn

hang).

Contents lists available at ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

://dx.doi.org/10.1016/j.vetmic.2013.11.005

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271262

and small (S1–S4) (Nick et al., 1975; Spandidos andGraham, 1976; Gouvea and Schnitzer, 1982; Benaventeand Martınez-Costas, 2007). All avian reovirus (ARV)encoded proteins include at least 10 structural proteins(lA, lB, lC, mA, mB, sC, sA, and sB) and four nonstructuralproteins (mNS, p10, p17, and sNS).

Avian reoviruses have been associated with differentdiseases in a variety of domestic and wild birds, includingchicken (Olson and Weiss, 1972), goose (Palya et al., 2003;Yun et al., 2012), turkey (Simmons et al., 1972), Muscovyduck (Gaudry et al., 1972), Pekin duck (Jones andGuneratne, 1984), pigeon (Vindevogel et al., 1982), quail(Ritter et al., 1986), psittacine birds (Conzo et al., 2001),and several other wild bird species (Heffels-Redmannet al., 1992; Kuntz-Simom et al., 2002). Birds are mostsusceptible at a young age (Rosenberger et al., 1989).

Classical Muscovy duck reovirus (MDRV) is theetiological agent of a disease first described in SouthAfrica in 1950 (Kaschula, 1950) and then isolated in Francein 1972 (Gaudry et al., 1972). MDRV mainly infectsducklings between 2 and 4 weeks of age; their resultingmorbidity is high, and their rate of mortality ranges from10 to 50%. In addition, recovered Muscovy ducks aremarkedly stunted in growth. The disease is characterizedby general weakness, diarrhea, serofibrinous pericarditis,swollen liver and spleen, and covered small white necroticfoci (Gaudry et al., 1972; Malkinson et al., 1981; Pascucciet al., 1984; Marius-Jestin et al., 1988).

MDRV shares common properties with avian reovirus,such as syncytium formation in cell culture and inability tohamagglutinate (Malkinson et al., 1981). However, severalnotable differences exist between MDRV and ARV,including different antigenicity by cross-neutralizationtests (Heffels-Redmann et al., 1992), host species differ-ences (chicken and Muscovy duck), pathogenic properties(Marius-Jestin et al., 1988), protein profiles (Heffels-Redmann et al., 1992), electropherotypes, and genomiccoding assignments (Kuntz-Simom et al., 2002). Forexample, the classical MDRV minor outer capsid proteinsC is encoded by S4 (Kuntz-Simom et al., 2002) and not byS1, as is usually found in ARV.

In China, classical MDRV infection has been reportedsince 1997 (Wu et al., 2001). The virus isolates shareidentical properties in pathology, culture, and genome(Kuntz-Simom et al., 2002; Zhang et al., 2007; Banyai et al.,2005; Wang et al., 2013). Since 2002, a new infectiousdisease emerged among Muscovy ducks and geese inSoutheast China. The disease is characterized mainly byhemorrhagic-necrotic lesions in the liver and spleen of thesick birds and is tentatively designated hemorrhagic-necrotic hepatitis (Liu et al., 2011). Recently, the causativeagent of the disease was isolated and identified; itspathogenicity, growth properties, and genome sequencesclassify it as a novel duck reovirus (NDRV) (Chen et al.,2012; Yun et al., 2012; Ma et al., 2012; Wang et al., 2012).

In this study, a novel duck reovirus strain, named N-MDRV ZJ00M, was isolated from a diseased Muscovyduckling in Zhejiang province of China in 2000. Its wholegenome was cloned, sequenced, and analyzed. The genomeof N-MDRV ZJ00M exhibited distinct molecular character-istics compared with ARV and classical MDRV. The study

revealed that N-MDRV has existed in China at least sincethe 2000s and provided additional insights into thereassortment and evolutionary relationship within intra-and interspecies of Orthoreovirus species groups II.

2. Materials and methods

2.1. Virus isolation and virological characterization

Liver samples of the dead Muscovy duckling withhemorrhagic-necrotic lesions were collected from a duckfarm in Zhejiang Province and processed for virus isolationusing embryonated SPF chicken eggs. Briefly, the liversamples were homogenized in PBS (pH 7.2) containingantibiotics (10,000 units/ml penicillin and 10,000 mg/mlstreptomycin) to obtain a 20% suspension (w/v). Thesuspension was centrifuged at 12,000 � g for 10 min andthen inoculated on the chorioallantoic membrane of 10-day-old chicken embryos (0.2 ml/embryo). The embryonicviability was monitored daily for 7 days. For cell culturepassage, the allantoic fluid was inoculated into chickenembryo fibroblast (DF-1) cells (1.0 ml of a 1:10 dilution inmedium) and incubated at 37 8C for 1 h for virusadsorption. The inoculums were then removed, and freshmedium containing 1% FBS was added. The cells wereincubated for an additional 48–72 h at 37 8C/5% CO2 andchecked daily for cytopathic effects (CPE). The cells werefreeze-thawed three times, the cellular debris wasremoved through low-speed centrifugation, and thesupernatant fluid was stored at �70 8C for the followingexperiments. The isolated virus was cultured for at leastthree passages for amplification and sequencing.

The virus titers were determined by plaque assay onDF-1 cells (Igarahi et al., 1981; Okuno et al., 1984). Briefly,monolayer cultures of DF-1 cells (1 � 105/well) grown insix-well plates were incubated with 10-fold serial dilutionsof the virus for 1 h at 37 8C. The infected cells were thenoverlaid with 2 ml of DMEM containing 1.5% methylcellulose and 2% fetal bovine serum and incubated at 37 8Cunder a 5% CO2 atmosphere for 72 h. The cells were fixedwith 1 ml of 10% formaldehyde for 30 min, washed withPBS (pH 7.2), and stained with methylene blue tetrahy-drate solution to visualize the plaques, and the visualizedplaques were counted.

The virions were purified by differential centrifugation.First, the virus suspension (crude extract) was centrifugedat 10,000 � g for 30 min at 4 8C to remove the cellulardebris. Second, the resultant supernatant was precipitatedwith 50% saturated ammonium sulfate at 4 8C. Theprecipitate was collected by centrifugation at 10,000 � g

for 20 min and suspended in a buffer consisting of 0.02 MTris (pH 7.0), 0.001 M EDTA, and 0.15 M NaCl. This virusbuffer was then ultracentrifuged for 3 h at 130,000 � g in aBeckman SW70 rotor at 4 8C on a 40% sucrose cushion (W/V, prepared with PBS), and the virus pellet was resus-pended in 50–100 ml of cold DEPC H2O and stored at�80 8C until use.

The viral morphology was determined through trans-mission electron microscopy as described previously(Hoshino et al., 2007). Briefly, the cells were fixed in2.5% glutaraldehyde and 1% osmic acid for 2 h on ice, and

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T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271 263

cell pellets were dehydrated and embedded in Epon (Nissin EM, Tokyo, Japan). Thin sections were cut,ned with uranyl acetate, and examined with a Hitachidel H-7000 transmission electron microscope. Theified virus particles were placed onto Formvar-bon-coated copper grids, negatively stained with 2%sphotungstic acid (PTA), 2% uranyl acetate (UA), or UA- double staining, and examined with an electronroscope.

The dsRNA from the virus was extracted using themercial TRI-Reagent (TaKaRa) according to the man-

cturer’s instructions. It was then precipitated with 2 Ml to remove the ssRNA. The dsRNA was purified from theernatant using a column from the Qiagen Gel Extrac-

kit. The extracted RNAs were maintained at �70 8C. viral dsRNA were analyzed by SDS-PAGE on vertical

gels (7.5% polyacrylamide gel) in Laemmli’s discon-ous buffer (Laemmli, 1970). The RNA segments werealized by silver staining (Dolan et al., 1985).

A method of full-length amplification of cDNA (FLAC),ich was optimized and described by Maan et al. (2007)

Potgieter et al. (2009), was used to obtain the entireuences of the genomic segments of N-MDRV. A hairpinhor primer, FLAC loop (50-p-GACCTCTGAGGATTC-AC TCCAGTTTAGAATCC. . .OH-30), which is similar to

t described by Maan et al. (2007), was ligated to thel dsRNA. The ligation reaction was performed by T4

A ligase (New England Bio Labs, UK). The ligated dsRNAs purified using agarose gel extraction columns follow-

the manufacturer’s recommendations (TaKaRa). Thet strand cDNA of the genome segments was synthesizedng an M-MLV reverse transcription system (Promega,

) according to the recommended protocols. The cDNAs used directly for PCR or stored at �20 8C. Theplification of the cDNA was performed using a 50

sphorylated FLAC 2 primer (50-CCGAATTCAGTTTA-ATCCTCAGAGGTC-30), which contains the restrictionyme sites for EcoRI (underlined) to facilitate the cloning

subcloning of the amplified cDNA. To ensure that theleotide sequences did not contain PCR-based errors,

ee clones of each gene of DRV were sequenced.

equencing and sequence analysis

The amplified cDNA products were separated on a 1% agarose gel, and the individual segments were purified

ng an agarose gel extraction kit. The purified PCRducts were cloned into T/A cloning vectors pMD-Simple (TaKaRa). The positive clones were identifieded on the size of the inserts. The terminal ends of theA inserts were all sequenced with M13 universalers using an ABI PRISM Big Dye Terminator Cycle

uencing Ready Reaction kit (version 2.0) on an ABISM 3730 DNA sequencer (Perkin-Elmer Applied Bio-tems). When necessary, custom primers were designedbtain the complete sequence of both strands of each

ert.The sequence analysis was performed using the Clustalsoftware program (Thompson et al., 1994) and MEGA

(Tamura et al., 2011). Phylogenetic trees based on theleotide sequences of all 10 genome segment were

constructed using the maximum likelihood method(Tamura et al., 2011) with 1000 bootstrap iterations. Thenucleotide sequence data reported in this study have beendeposited in the GenBank database and have been assignedaccession numbers for each gene of NDRV.

4. RT-PCR assays of diseased duckling and goslingsamples

Diseased ducklings/goslings with hemorrhagic-necro-tic symptoms and white necrotic foci were collected fromdifferent farms in Zhejiang, Fujian, and Jiangsu provincesduring 2000–2013. The total RNA from the liver or spleenof the diseased birds was extracted according to themanufacturer’s protocol and used as templates for RT-PCR.Specific primers for the Sigma C gene were designed andsynthesized according to the S1/S4 segments of N-MDRVZJ00M and classical MDRV (AY580259). (N-MDRV: sC-F:50-ACGATGGATCGCAACGAGGTG-30, sC-R: 50-GATGAA-TAGCTCTTCTCATCGC-30; classical MDRV: sC-F: 50-CCTGGAACGAATACCACCTTCA-30, sC-R: 50-CAAATGGTCG-CAATGGAGAAGC-30). The RT-PCR products were purifiedand sequenced, and their lengths were 1001 bp (N-MDRV)and 826 bp (classical MDRV), respectively.

5. Results and discussion

5.1. Virus culture, electron microscopy, and SDS-PAGE

analysis

Seven days after chorioallantoic membrane inoculationof the liver homogenate, all of the six SPF chicken embryoshad died. The embryo allantoic fluid collected from the firstpassage caused 100% mortality of the chicken or duckembryos in the subsequent passages. Both embryosexhibited edema with severe subcutaneous hemorrhageand multiple necrotic foci in the liver and spleen. Theinoculation of monolayers of DF-1 cells resulted in thedevelopment of CPE, usually after 3–5 blind passages. FocalCPE appeared with the cells rounding up and floating freefrom the surface of the flask 72 h post infection (Fig. 1A).Fusion of the infected cells and formation of syncytium,which is characteristic of ARVs, was not observed in any ofthe TC systems. No hemagglutination activity was detectedin the allantoic fluid and cell culture supernatants. Theviral titer determined at three days post-infection was106.5 TCID50/ml. The electron microscopy observationsrevealed that the virus displayed a spherical shape andpossessed two capsid layers. In addition, the virus was75 nm in diameter and thus similar in size to known ARVs,which range from 70 to 90 nm in size. The boundarybetween the outer capsid and the inner core is evident, asshown by a prominent white ring in the negatively stainedelectron micrograph (Fig. 1B).

The N-MDRV ZJ00M genomes were purified andanalyzed by SDS-PAGE. As shown in Fig. 1C, the genomesegments were separated into 10 distinct bands, withsegments 1 and 2 co-migrating. The comparison of thegenomes of N-MDRV ZJ00M, classical MDRV, and ARV-S1133 showed that all of the segments of each segmentgroup (large, L; medium, M; small, S) display some

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271264

difference in their electrophoretic mobility (Fig. 1C). The N-MDRV ZJ00M, classical MDRV, and ARV L and M genesmigrated to similar positions in the gel, and majordifferences were clearly evident in the mobility of the Ssegments. The analysis of the N-MDRV ZJ00M segmentrevealed that the S1 gene position migrates more closely tothe M segments than to the other S genes, whereas the fourclassical MDRV S genes migrated together, and the other Sgenes (S2–S4) positions are similar to the genes of ARV-S1133.

6. The genome of N-MDRV ZJ00M

The complete sequences of segments 1–10 of N-MDRVZJ00M were obtained and have been deposited in GenBankunder accession numbers KF154110 to KF154119. Thecomplete genome sequence of N-MDRV ZJ00M wasdetermined to consist of 23,419 bp divided in 10 segments

G+C content for each segment was 50.13% and varied onlyby �1.35% between the different segments (Table 1).Reovirus segments normally encode a single open readingframe (ORF) that spans almost the entire length of thesegment. However, there have been several cases wherereovirus segments encode more than one ORF (Bodelon et al.,2001; Mattion et al., 1991; Suzuki et al., 1996). Based onpredicted ORF start and stop codons, each DRV segment wasexamined to determine its open reading frames (ORFs). Withthe exception of the S1 genome segments, which containthree ORFs, the other segments contained only one ORF. TheORFs of the S1 genome have a partially overlapping genearrangement and consist of the p10, p18, and sC ORFsarranged from 50 to 30 on the S1 genome segment plus strand(mRNA) (Table 2).

Each N-MDRV ZJ00M segment sequence was used as aquery in BLASTp searches of the non-redundant proteindatabase. Several ARV and classical MDRV proteins

Fig. 1. Cytopathic effect, electron micrograph, and genome electrophoresis of N-MDRV. (A) Cytopathic effect (CPE) induced by N-MDRV in DF-1cell lines

(10 � 20). (B) Electron micrograph of the purified isolate negatively stained with 2% phosphotungstic acid. Note the double capsid structure. The scale bar

represents 50 nm. (C) Comparison of the electrophoretic mobility of N-MDRV, classical MDRV and ARV genome segments. Purified genomic dsRNA

segments of ARV-S1133 (lane 1), classical MDRV (lane 2) and N-MDRV ZJ00M (lane 3) were analyzed by 7.5% PAGE and visualized by silver staining. The

locations of the large (L), medium (M) and small (S) size class segments are indicated, along with the numbering scheme of the S class genome segments.

showed the highest amino acid identity to the putative

that range in size from 1191 bp to 3959 bp. The average

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T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271 265

DRV ZJ00M orthologs, including ORFs L1, L2, L3, M1,, M3, S1a, S1b, S2, S3, and S4. Therefore, the putativetein functions of the respective ORFs were proposeded on their homology to their ARV counterpartsble 1).

on-coding regions of N-MDRV ZJ00M genomements

As shown in Table 1, the 50- and 30-non-coding regionsRs) of the segments were short, averaging 20 nt and 59respectively. Conserved terminal nucleotide sequencese been considered as a feature in reovirus classification. comparison of the genome sequences of DRV showedt all of the segments contained conserved terminaluences. As is typical of the family Reoviridae (Mertens

Sangar, 1985; Kudo et al., 1991; Isogai et al., 1998a), terminal sequences of N-MDRV ZJ00M positive-senseAs were conserved among the segments. All 10 N-MDRV0M segments shared the 50-GCUUUUU motif at the 50-

and the 50-UCAUC motif at the 30-NCR. The 50-NCRs 30-NCRs contained motifs that were highly conservedpared to the ARVs.

The conserved nucleotides 50-GCUUUUU-30 were pre-t at the 50 ends in all of the positive strands of eachment, and 50-UAU/CUCAUC-30 was present at the 30

s. These were very similar to that found in group II of Orthoreovirus genus, which includes ARV (50-

UUUU-30 at the 50 end and 50-UAUUCAUC-30 at 30

) and classical MDRV (50-GCUUUUU-30 at the 50 end andAU/CUCAUC-30 at the 30 end; Table 3). Conservedinal sequences can be used in genome assembly and

kaging as ‘‘sorting’’ signals (Xu et al., 1989).Moreover, the first and last nucleotides of each segmentll orthoreoviruses were complementary (G-C). Poten-

imperfect inverted repeats were also predicted in theuences adjacent to each termini of the N-MDRV ZJ00M

positive-sense strand (Table 4). It has been reported thatcomplementary sequences in the 50- and 30-NCR mayfacilitate viral replication by circularizing the RNAtranscript (Patton, 2001).

Orthoreovirus genome segments characteristicallyhave non-coding regions at the 50 and 30 ends of eachcoding strand. In N-MDRV, these nucleotide sequences arenot conserved between the 10 genome segments, and theregions at the 50 end are consistently smaller in length (12–30 nucleotides) compared to those at the 30 end (32–98nucleotides). These features have previously been reportedfor the genome segments of the MRV, ARV, NBV, and BRVspecies (Duncan, 1999).

8. Comparison with other orthoreovirus species

Orthoreoviruses share a wide range of nucleotide (nt)and amino acid (aa) sequence identities. The extent ofsimilarity has served as a basis for the classification oforthoreoviruses into different groups (I–V) (Mertens et al.,2000). The nt and aa sequence identities are greater than75% and 85% between homologous orthoreovirus genes,which results in the strains being classified into the samespecies group. In contrast, if the nt and aa identities are lessthan 60% and 65%, respectively, the strains belong todistinct species group (Mertens et al., 2000).

The genes and proteins of N-MDRV ZJ00M werecompared with their homologs from other orthoreovirusspecies (Table 2). The results showed that N-MDRV ZJ00Mhas a higher similarity (nt, 31.4–99.7%; aa, 14.1–100%)with the avian orthoreoviruses (ARV, classical MDRV andN-MDRV etc.) in Orthoreovirus genus, particularly with N-MDRV (nt, 86.8–99.6%; aa, 94.6–100%). For ZJ00M andARVs (ARV and classical MDRV), the sequence comparisonsof the 10 genome segments showed that the sequencedivergences of the outer capsid protein (mB, sB, and sC)-encoding genes were significantly higher than those of

le 1

racteristics of genome segments and predicted functions of proteins in novel Muscovy duck reovirus (N-MDRV).

nome

gment

Gene Protein Predicted functiona

Segment

length (bp)

GC% 50UTR

(bp)

30UTR

(bp)

ORF

coordinates

Coding

potential

Protein

size (aa)

MV

(KDa)

Isoelectric

point (PI)

3959 49.79 21 56 22–3902 lA 1293 142.2 6.307 Inner core protein, core–shell

scaffold

3830 48.93 14 36 15–3793 lB 1259 139.9 8.042 Inner core protein, putative

transcriptase (RdRp)

3907 48.61 12 37 13–3870 lC 1285 142.0 5.059 Turrets, capping enzyme

1 2284 49.30 12 73 13–2211 mA 732 82.1 8.297 Inner core, putative transcriptase

co-factor

2 2158 50.97 29 98 30–2060 mB 675 73.1 5.106 Outer capsid protein, penetration

3 1996 52.05 24 64 25–1932 mNS 635 70.7 6.303 Nonstructural protein, formation

of viral factories and protein

recruitment

1568 49.59 19 32 273–761 P10 97 10.2 6.554 Nonstructural protein,

permeabilising/fusogenic

42.86 571–1536 P18 162 18.3 8.283 Nonstructural protein, unknown

51.33 20–313 sC 321 34.1 5.995 Outer capsid, cell attachment

1324 52.04 15 58 16–1268 sA 416 46.1 8.544 Inner core protein, dsRNA binding

1202 49.42 30 68 31–1134 sB 367 41.4 6.630 Outer capsid protein, unknown

1191 51.39 23 64 24–1127 sNS 367 40.1 6.825 Nonstructural protein, ssRNA

binding

Protein coding assignments and functions proposed by Benavente and Martınez-Costas (2007).

Table 2

Percent sequence identities of genome segments and proteins between N-MDRV and orthoreovirus species.

Orthoreovirus lA lB lC mA mB mNS sA sB sC/s1 p10a p18/NSPa sNS

nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa

ARV 138 77.0 94.7 77.2 91.3 70.1 79.4 73.6 86.3 77.4 90.4 71.5 80.2 77.5 91.8 59.6 68.1 38.8 28.5 46.9 34.0 31.8 14.1 78.1 90.7

176 77.6 94.6 75.8 91.5 70.1 79.3 73.6 86.2 76.8 89.0 71.7 79.8 77.5 91.3 60.1 69.2 38.4 29.7 47.6 33.0 31.4 14.1 77.9 90.5

S1133 77.6 94.7 75.7 91.2 70.3 79.5 73.6 86.2 76.7 89.8 71.8 80.3 77.1 90.4 59.8 68.9 38.3 29.4 48.3 33.0 31.6 14.1 78.0 89.4

MDRV 815–12 85.8 97.4 87.9 98.0 79.7 93.0 95.6 96.9 67.3 75.6 85.9 94.2 87.5 98.1 61.1 68.7 52.9 41.6 35.9 7.9 NE NE 84.6 97.0

S14 NA NA NA NA NA NA 81.2 94.4 67.4 76.3 86.2 94.2 87.6 98.1 61.7 69.2 53.1 42.0 NA NA NE NE 85.1 97.3

N-MDRV 03G 86.8 98.0 98.2 97.5 96.8 98.6 96.4 97.0 87.9 96.1 94.8 96.4 88.8 97.6 94.7 94.6 97.4 97.5 98.3 100.0 96.7 95.7 94.3 97.8

091 97.8 99.2 97.5 99.7 97.6 99.1 99.1 99.0 98.5 99.0 99.4 99.1 89.0 98.3 97.5 97.5 97.9 98.4 98.6 100.0 98.4 100.0 99.4 100.0

J18 97.1 99.6 88.0 99.0 98.4 99.3 97.5 98.5 97.9 99.0 99.4 99.2 99.6 100.0 97.7 96.2 97.4 97.2 98.0 100.0 97.3 96.9 93.8 97.0

BRoV 53.4 50.9 54.2 51.2 40.7 26.1 44.1 33.8 50.0 46.3 39.2 21.9 45.2 34.6 25.1 21.2 NA NA 35.5 14.3 30.6 4.9 45.0 35.6

BRV 54.4 51.6 52.9 50.2 40.6 25.8 44.5 32.2 47.0 38.5 36.9 21.6 40.0 28.7 24.7 19.7 NA NA 35.2 10.1 27.4 7.7 41.7 27.4

NBV 66.2 72.6 64.7 70.9 47.5 40.5 52.1 46.9 64.2 68.5 48.5 39.1 58.7 59.6 32.0 32.9 32.7 23.3 43.1 31.9 31.9 16.2 54.1 51.0

Pulau 66.7 73.6 58.1 71.0 47.9 40.6 52.8 47.2 63.0 68.5 48.5 38.9 56.8 59.1 32.5 31.8 32.2 18.9 44.4 30.9 32.5 15.8 54.1 51.0

RRV NA NA NA NA NA NA NA NA NA NA NA NA NA NA 28.2 26.3 30.8 17.8 32.8 12.8 NE NE NA NA

MRV MRV1 50.6 43.8 54.2 53.7 41.8 27.8 43.5 27.3 51.4 45.5 42.3 22.7 41.9 28.2 24.2 17.7 31.7 17.1 NE NE 43.9 11.9 42.7 23.8

MRV2 49.4 44.1 54.9 54.0 40.6 27.4 43.5 27.4 51.6 45.3 43.1 23.2 42.0 28.2 22.5 17.7 31.7 15.9 NE NE 39.5 7.1 43.1 23.8

MRV3 50.6 43.8 54.2 53.5 41.0 27.5 43.2 27.0 51.4 45.5 42.7 22.1 42.7 28.2 22.2 17.5 32.6 15.9 NE NE 33.7 6.7 42.7 23.5

aa: amino acid sequence.

nt: nucleotide sequence.

NA: sequence not available.

NE: no equivalent sequence.a Nucleotide sequence of the polycistronic genome segment: the S1 genome segments of MRV, ARV, NBV and RRV species, the S4 genome segment of classical MDRV and BRV (and BroV) species.

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16

8 (2

01

4)

26

1–

27

12

66

othS1,

seqsegexeattabetlog

Tab

Con

the

Or

I.

II.

III

IV

V.

VI

Tab

Con

RN

L1

L2

L3

M

M

M

S1

S2

S3

S4

Co

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271 267

er genes. In addition, the tricistronic genome segmentwhich encodes sC, displayed the greatest level of

uence divergence among the L-, M-, and S-class genomements. This is likely due to the selective pressuresrted on the reovirion outer capsid, particularly the cellchment protein (sC). The overall identity values

ween N-MDRV ZJ00M and other orthoreovirus homo-ous proteins ranged from 6.7% to 73.6% (Table 2). Based

on the level of sequence identity between the genomesegments and encoded proteins of N-MDRV ZJ00M andother orthoreovirus species, N-MDRV ZJ00M was assignedto the avian orthoreoviruses species in the genusOrthoreovirus.

Furthermore, segment sequence analysis and align-ment revealed that the functional domains, conservedamino acid residues, and sequence motifs of N-MDRVZJ00M in the l-, m-, and s-class proteins are almostidentical with those previously identified in the homo-logous proteins of ARV and classical MDRV (Liu et al., 2003;Noad et al., 2006; Su et al., 2006; Xu and Coombs, 2008;Kuntz-Simom et al., 2002; Banyai et al., 2005; Zhang et al.,2007; Wang et al., 2013), which suggests that thefunctional domains, conserved amino acid residues, andsequence motifs are conserved in the genome segments ofARV, classical MDRV, and N-MDRV.

9. Phylogenetic analysis

The evolutionary relationships of N-MDRV ZJ00M withthe Orthoreovirus genus members, particularly ARV and

le 3

served terminal nucleotide sequences on dsRNA genome segments of

six recognized orthoreovirus species groups and N-MDRV are listed.

thoreovirus species group Conserved terminal

nucleotide sequences

Mammalian orthoreovirus

(MRV)

50-GCUA. . .UCAUC-30

Avian orthoreovirus (ARV) 50-GCUUUUU. . .UAUUCAUC-30

Moscovy reovirus (MDRV) 50-GCUUUUU. . .UAU/CUCAUC-30

Novel duck reovirus (N-MDRV) 50-GCUUUUU. . .UAU/CUCAUC-30

. Nelson bay virus (NBV) 50-GCUUUA. . .UCAUC-30

. Baboon orthoreovirus (BRV) 50-GUAAA. . .UCAUC-30

Reptilian orthoreovirus (RRV) 50-GUUAUUUU. . .UCAUC-30

. Broome virus (BroV) 50-GUCAA. . .UCAUC-30

le 4

served terminal sequences and imperfect inverted repeats (shaded areas) located at both termini nucleotide sequences of N-MDRV genome segments.

N-MD RV termina l s equenc es

A s egment 5' end 3' end

Inverted repea ts

GCUUUUUCUCC GAACG CGUU GGAGGUUAUUCAUC

GCUUUUUCCUC ACC AUGC GCAUGGCUCGAG GAAUUACUC AUC

GCUUUUU CACCC GGGUGUUACUCAUC

1 GCUUUUUCU CGAC GUC UUGA GAUAUUCAUC

2 GCUUUUUGA GUGCUAA UUGGCACGUUAUUCAUC

3 GCUUUUU GAGUCC GGAC UCGGUUAC UCAUC

GCUUUUUCU UCUC GAGAA GAGCUAUUCAUC

GCUUUUUCUCC CACG CGUGGGUGUAUUCAUC

GCUUUUU GAGUCC GGAC UCGCC UAUUCAUC

GCUUUUU GAGUCC GGAC UCUUAUUCAUC

nsensus GCUUUUU UC AU

C

Fig. 2. Phylogenetic trees built based on nucleotide sequences of the homologous L, M and S genome segments of Orthoreovirus species, using maximum

likelihood method in the mega 5 program (Tamura et al., 2011). Bootstrap values of 1000 replications are shown at the notes. All trees are plotted to the

same scale. The bar indicates genetic distance. (^) Strain determined in this study.

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271268

classis.imunucall

resdesthethegro(FignucoutwaJ18whrec815anotheet afurtMDgrophyof lphyprogen

Tab

The

Sa

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

Po

WN

‘‘+’’:

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271 269

sical MDRV, were determined by phylogenetic analy- Phylogenetic trees were constructed using the max-m likelihood method with bootstrapping based on theleotide sequences of all 10 genome segments (Fig. 2). Inof the phylogenetic trees, each case yielded similar

ults: the N-MDRV ZJ00M is distinct from all othercribed reovirus species groups but still classified into

previously defined Orthoreovirus species group II with other ARVs (including DRV and GRV) and furtheruped into the classical MDRV and GRV genogroup. 2). The phylogenetic trees constructed using theleotide sequences of the segments encoding the threeer capsid proteins (mB, sB, and sC) showed that ZJ00Ms classified into the cluster containing N-MDRV (091,, TH11, and NP03 strains) and N-GRV (03G strain),ich are the Chinese reovirus strains that emerged inent years, and classical MDRV and GRV (including the–12, S14, 89026, 89330, and D14/99 strains) constitutether cluster within the MDRV genogroup. According to

criteria used for the genotype classification of ARV (Liul., 2003; Su et al., 2006), the MDRV genogroup could beher divided into two different genotypes. The classicalRV and GRV belong to genotype 1, and N-MDRV isuped into genotype 2. In addition, the results of thelogenetic analysis based on the nucleotide sequencesC and sNS was identical to those obtained from thelogenetic analysis based on the three outer capsidteins: the MDRV genogroup can be divided into twootypes.

Viral RdRp is an important gene for the phylogeneticanalysis of Reoviridae, and phylogenetic analysis based onthe gene sequence of RdRp has been used to confirm theevolutionary status of various species (Attoui et al., 2006;Mohd Jaafar et al., 2008; Stenger et al., 2009; Allyn et al.,2012). In phylogenetic trees based on the nucleotidesequences of RdRp, lA, mA, and mNS, the N-GRV 03Gisolate was more closely related to the classical MDRV815–12 isolate than to other N-MDRV isolates.

Based on the topological heterogeneity observedamong the phylogenetic trees, we hypothesize that agenetic reassortment of the L, M, and S segments likelyoccurred within Orthoreovirus species group II.

10. RT-PCR samples of different years and regions

Total RNA was extracted from diseased duckling orgosling samples and used as templates for RT-PCR. Specificamplification bands (N-MDRV, 1001 bp; classical DRV,826 bp) were obtained from all of the samples. Thenucleotide sequence alignments of the amplificationproducts showed that 80.8% of the samples (23/26)displayed high nucleotide sequence similarities to thesC (S1) sequence of N-MDRV (97.3–99.7%). Only threesamples (white necrotic foci in the liver and spleen) weredetected by specific primers of classical MDRV, and theamplification products shared 96.1–97.8% identities withthe sC (S4) nucleotide sequence of classical MDRV(AY580259). The phylogenetic tree constructed based on

le 5

detection of N-MDRV and classical MDRV in the clinical samples by RT-PCR during 2000–2013.

mples Pathotype Districts Date collected Breeds Results

N-MDRV Classical MDRV

WNF Zhejiang 2000 Muscovy duckling � +

WNF Zhejiang 2000 Muscovy duckling � +

WNF Zhejiang 2000 Muscovy duckling � +

HNL Zhejiang 2000 Muscovy duckling + � HNL Zhejiang 2003 Gosling + � HNL Zhejiang 2005 Muscovy duckling + � HNL Zhejiang 2005 Muscovy duckling + � WNF Zhejiang 2006 Muscovy duckling + � HNL Zhejiang 2006 Wild duckling + � HNL Zhejiang 2008 Muscovy duckling + � HNL Fujian 2008 Muscovy duckling + � HNL Beijing 2009 Peking duckling + � HNL Zhejiang 2009 Wild duckling + � HNL Zhejiang 2010 Muscovy duckling + � HNL Jiangsu 2010 Muscovy duckling + � HNL Zhejiang 2010 Gosling + � HNL Zhejiang 2011 Muscovy duckling + � HNL Jiangsu 2011 Muscovy duckling + � HNL Zhejiang 2011 Muscovy duckling + � HNL Jiangsu 2012 Muscovy duckling + � HNL Zhejiang 2012 Muscovy duckling + � HNL Zhejiang 2012 Muscovy duckling + � HNL Jiangsu 2012 Muscovy duckling +

HNL Zhejiang 2012 Muscovy duckling + � HNL Zhejiang 2013 Muscovy duckling + � HNL Zhejiang 2013 Shaoxing duckling + �

sitive rate% (positive numbers/total numbers) 80.8%(23/26) 19.2%(3/26)

F: white necrotic foci; HNL: hemorrhagic-necrotic lesions.

positive; ‘‘�’’: negative.

T. Yun et al. / Veterinary Microbiology 168 (2014) 261–271270

the segment encoding sC (Fig. 2) also revealed that the No.4-26 samples were grouped in the N-MDRV cluster, andthe No. 1-3 samples were classified into the classical MDRVgenotype. This finding suggests that N-MDRV has existedin China since 2000 and is a currently popular genotype insoutheastern China. In addition, different duck breeds andgeese, including Muscovy duck, Peking duck, Shaoxingduck, and domesticated wild duck, are affected by N-MDRV(Table 5).

11. Conclusions

In summary, N-MDRV exhibits a number of differentproperties and pathogenicity compared with ARVs (clas-sical MDRV and GRV). These differences mainly include thefollowing: N-MDRV causes disease in different duck breedsthat is mainly characterized by hemorrhagic-necroticlesions in the liver and spleen; N-MDRV does not causesyncytium formation in cell culture; the S1 segment of N-MDRV is a tricistronic gene that encodes the overlappingORFs for p10, p18, and sC. Phylogenetic analysis indicatesthat N-MDRV is distinct from all other described reovirusspecies groups (I–IV) but is still classified into theorthoreovirus species group II with the other ARV andclassical MDRV and is grouped into the MDRV genogroup.In addition, the MDRV genogroup can be further dividedinto two genotype clusters. Furthermore, the RT-PCRassays of samples from different regions and yearssuggests that N-MDRV has existed since 2000 and iscurrently a widely prevalent genotype in southeasternChina.

Acknowledgments

This work was supported by grants from the SpecialFund for Agro-scientific Research in the Public Interest(201003012), Public Welfare Technology Research Projectof Zhejiang Province (2012C22074), the Zhejiang NaturalSciences Foundation (LY13C180002), Three Rurals and SixParties Project of Zhejiang Province and the Scientific andTechnological Innovation Team of Zhejiang Province.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.11.005.

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