Ao

YS

a

ARRAA

KM�PE

1

gp(mSi

ac(daimd1

H1

(

0d

Virus Research 163 (2012) 546– 551

Contents lists available at SciVerse ScienceDirect

Virus Research

journa l h o me pag e: www.elsev ier .com/ locate /v i rusres

ntigenic analysis monoclonal antibodies against different epitopes of �B proteinf Muscovy duck reovirus

ongfeng Li, Xiuchen Yin, Xiaodan Chen, Xiaojun Li, Jinzhe Li, Chunguo Liu, Ming Liu ∗, Yun Zhang ∗

tate Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, PR China

r t i c l e i n f o

rticle history:eceived 28 September 2011eceived in revised form 7 December 2011ccepted 8 December 2011vailable online 16 December 2011

eywords:uscovy duck reovirus (DRV)B protein

a b s t r a c t

�B is one of the major structural proteins of Muscovy duck reovirus (DRV), which is able to induce pro-tective immune response in target birds. Four anti-DRV �B MAbs were identified belong to two distinctepitopes, designated A (1E5, 4E3, and 5D8) and B (2F7) (Liu et al., 2010). To understand antigenic deter-minants of the �B protein, a set of 20 (P1–P20), partially overlapping and consecutive peptides spanning�B were expressed and then screened by MAbs. With Western blot and enzyme-linked immunosorbentassay (ELISA), two minimal units of the linear epitopes, 19YIRAPACWD27 (epitope B) and 65TDGVCF-PHHK74 (epitope A), were identified within N-terminal region of the �B protein. The epitope B washighly conserved among DRV and avian reovirus (ARV) strains through sequence alignment analysis.

epscanpitope

Immunofluorescence assays (IFA) and ELISA, confirmed that epitope B is a broad group-specific epitopeamong DRV and ARV. Epitope A could only react with chicken embyonated fibroblast cells (CEF) infectedwith DRV, but not ARV. However, both peptides have good immunogenicity and could induce antibodiesagainst DRV in BALB/c mice. This report documents the first identification of �B epitopes in the preciselocations. The two probes would be useful in the development of discriminating diagnostic kits for DRV

and ARV infection.

. Introduction

Muscovy duck reovirus (DRV), a member of the Orthoreovirusenus of the Reoviridae family, is an important aquatic birdathogen. DRV consists of ten segments of double stranded RNAdsRNA) that can be separated into three size classes, large,

edium, and small, based on electrophoretic mobility (Kuntz-imon et al., 2002; Zhang et al., 2007a). DRV encoded proteinsncluding at least ten structural and four nonstructural proteins.

Muscovy duck reovirus was first described as the etiologicalgent of disease in South Africa in 1950 (Kaschula, 1950). Despiteommon properties shared between duck and chicken reovirusnamed avian reovirus, ARV), the two viruses are antigenicallyifferent (Heffles-Redmann et al., 1992; Kaschula, 1950). DRV isn important pathogen associated with several diseases, includ-ng viral arthritis/tenosynovitis, growth retardation, pericarditis,

yocarditis, enteritis, hepatitis, respiratory syndromes, and sud-en death (Robertson and Wilcox, 1986; Rosenberger and Olson,991; McNulty, 1993). DRV could cause high morbidity and up

∗ Corresponding authors at: State Key Laboratory of Veterinary Biotechnology,arbin Veterinary Research Institute of CAAS, No. 427 Maduan Street, Harbin50001, China. Tel.: +86 451 85935067; fax: +86 451 82733132.

E-mail addresses: liuming04@126.com (M. Liu), yunzhang03@yahoo.comY. Zhang).

168-1702/$ – see front matter © 2011 Published by Elsevier B.V.oi:10.1016/j.virusres.2011.12.006

© 2011 Published by Elsevier B.V.

to 50% mortality in young ducks (Malkinson et al., 1981; Heffles-Redmann et al., 1992) and recovered ducks are normally stunted ingrowth.

Recently, several methods have been developed for the diag-nosis of DRV infections. An Enzyme-linked immunosorbent assay(ELISA) (Zhang et al., 2007b) is designed to detect antibody againstDRV. One tube PCR method has been developed as well for thedetection of DRV, ARV, and goose reovirus (GRV) RNA from the liverspecimens of infected birds or from cell cultures and allantic fluids(Zhang et al., 2006), providing a sensitive tool for the laboratorydiagnosis of these virus infections.

The �B of DRV, a major outer capsid protein, is structurallyrelated to the �3 protein of mammalian reovirus (MRV) and �Bof ARV and our data suggested it is functional related (Zhanget al., 2007b; Le Gall-Recule et al., 1999; Vakharia et al., 1996). The�B protein of DRV comprised 367 amino acids and could inducegroup-specific neutralizing antibody (Le Gall-Recule et al., 1999;Wichramasinghe et al., 1993), and its antigenic homology amongmany different ARV serotypes has been well documented. The DRVand ARV �B proteins contain conserved immunogenic regions, sug-gesting that �B protein can potentially be used as a target to raisepan-DRV/ARV �B MAbs for the detection of a broad spectrum of

birds’ reoviruses. Efforts have been made into mapping the epi-topes of � class proteins, including �A and �NS (Hou et al., 2001;Huang et al., 2005). However, the �B protein epitope antigenicityinformation is still not defined.

earch 1

wtotieb

2

2

w1p

2

aSfp3iwa

2

an(fept

2

(tdetocTWtT

2

efia3dn

Y. Li et al. / Virus Res

Four MAbs directed against bacterially expressed �B of DRVere developed and at least two distinct epitopes were existed on

he �B (Liu et al., 2010). To identify the precise locations of epitopesf MAbs, partially overlapping and consecutive peptides spanninghe �B were screened by MAbs. The two linear epitopes have goodmmunogenicity and reactogenicity. Antigenic analysis with het-rologous DRV and ARV isolates indicated that the two epitopeselong to group- and serotype-specific epitopes, respectively.

. Materials and methods

.1. Virus strains and monoclonal antibodies

DRV S12, S14, ARV S1133, and 99G strains used in this studyere described (Zhang et al., 2007b) and four �B-specific MAbs

E5, 4E3, 5D8, and 2F7 against the S12 strain of DRV were describedreviously (Liu et al., 2010).

.2. Broad epitope mapping with overlapping �B fragments

To localize the epitopes A and B on �B protein, a series ofmino- and carboxyterminal-deleted fragments of the �B of DRV12 strain were synthesized by PCR. Four partially overlappingragments (named P2–P5) spanning the �B was amplified fromET30a-�B (Zhang et al., 2007b) and then cloned into the pET-0a. The restriction enzyme Bam HI/Hind III sites were introduced

nto the corresponding primers (Table 1). The recombinant proteinsere expressed and then screened by Western blotting and ELISA

s described previously (Liu et al., 2010).

.3. Further limit the epitopes

Based on the epitope analysis using four peptides, P2, P3, P4,nd P5, 1–83 aa region of �B was determined to be the domi-ant antigen region of epitopes. Six partially overlapping fragmentsnamed P6–P11) spanning the 1–83 aa region of �B was amplifiedrom pET30a-�B and then cloned into pGEX6p-1. The restrictionnzyme Bam HI/Xho I sites were introduced into the correspondingrimers (Table 1). The recombinant proteins were expressed andhen screened by Western blotting and ELISA.

.4. Precise locations of the epitopes

To further map epitopes of �B precisely, two sets of threenamed P12–P14) and six (P15–P20) partially overlapping pep-ides spanning 65–85 aa and 15–44 aa of �B-encoding gene wereesigned, respectively. The complementary oligonucleotide pairsncoding each peptide were synthesized. After directly annealing,he sticky-ends of Bam I and Xho I sites were formed at the terminif the nucleotide segments. Then the nucleotide segments wereloned into pGEX6p-1 digested by the same restriction enzyme.he recombinant proteins were expressed and then screened byestern blotting and ELISA. The positions of the nine peptides and

he synthesized complementary oligonucleotide pairs are listed inable 1.

.5. SDS-PAGE and Western blotting

To localize the epitopes on �B of S12 strain, SDS-PAGE and West-rn blotting were performed. Purified �B, a series of truncated �Busion proteins, and protein extracts of the pET30a or pGEX6p-1nduced recombinant cells (as negative control) were mixed with

n equal volume of reducing Laemmli sample buffer, boiled for

min, and separated by SDS-PAGE. The gels were stained by stan-ard methods using Coomassie brilliant blue or electroblotted ontoitrocellulose (NC) membranes. NC membranes were probed with a

63 (2012) 546– 551 547

1:1000 dilution of MAbs followed by reaction with HRP-conjugatedgoat anti-mouse immunoglobulin (1:500 dilutions) (KPL, MD, USA).

2.6. Detection of the immunogenicity of epitope peptides

Female BALB/c mice were immunized subcutaneously on the1st and 15th days with 50 �g purified epitope peptides (P12, andP20) emulsified with complete and incomplete Freund’s adjuvants(Sigma, St. Louis, MO, USA), respectively. Ten days after the secondinjection, the sera were collected from mice. The antibodies againstP12 or P20 were detected by ELISA and Western blot.

2.7. Detection of the reactogenicity of the epitope peptides

To investigate whether the epitopes could be recognized by duckanti S12 or mice anti DRV �B sera, the epitope peptides P12 andP20 were used to coat the ELISA plates (Invitrogen) (50 ng/well) todetect the corresponding antibodies. Transformed into Escherichiacoli BL21 (DE3), the induced pET-30a or pGEX6p-1 empty vectorwas taken as negative control.

2.8. Homology analysis of the epitopes

Two epitopes sequences were analyzed by �B sequence align-ment of DRVs and ARVs. GenBank accession numbers for theARV/DRV �B-encoding genes were as follows: ARV S1133-�B,U20642; 1733-�B, AF004856; 2408-�B, AF301475; OS161-�B,AF301471; 918-�B, AF301473; 1017�B, AF301474; R2-TW-�B,AF301472; DRV 89330-�B, AJ243881; 89026-�B, AJ24388; S14-�B,EF100416.

2.9. Immunofluorescence assays

To further identify the specificities of MAbs 1E5 or 2F7 or miceanti P12 or P20 sera, ARV S1133 isolate was tested by indirectimmunofluorescence assays (IFA) as follows. Monolayers of CEFcells were infected with ARV S1133 strain and incubated for 48 h at37 ◦C. IFA was processed with MAbs or mice against P12 or P20 seraemployed as the primary antibodies and a FITC-conjugated goatanti-mouse IgG as the secondary antibody. Images were acquiredwith a Leica digital camera.

2.10. Cross-reactivity of MAbs to DRV and ARV strains

To study the MAbs 1E5 and 2F7 cross-reactivity to heterologousstrains, ARV S1133, 99G, and DRV S14 were used in an antigen-captured ELISA as described previously (Liu et al., 2010). On thebasis of cross-neutralization tests, DRV S12 and S14 or ARV S1133and 99G strains represent different serotypes. Mice polyclonal anti-body against DRV S12 and HRP-conjugated goat anti-mice antiserawere used as primary and secondary antibodies, respectively, todetect the presence of �B in each cell extracts. HRP-coupled MAbswere used directly as the primary antibodies. The relative bind-ing to heterologous isolates is expressed as a percentage by takingthe A405 obtained with DRV S12 in the reaction as 100. Bindingwas considered strong if it was more than 50%, significant if it was25–50%, and negative if it was less than 25%.

3. Results

3.1. Localization of the two epitopes of �B

The immunoreactivity of the peptides by Western blottingshowed that P1 (1–367 aa), P3 (1–181 aa), and P5 (1–83 aa) wererecognized by four MAbs 1E5, 4E3, 5D8 and 2F7 (Fig. 1A, a repre-sentative image), and no MAbs recognized P2 (174–367 aa) and P4

548 Y. Li et al. / Virus Research 163 (2012) 546– 551

Table 1Primers used for epitopes mapping.

Peptides Sequences of primers and synthesized oligonucleotides Position in �B (aa)

P1 5-TTTGGATCCAGCATGGAGGTACGTGTGC-3 (Bam HI) 1–3675-CCCAAGCTTCTACCAACCACACTCAATGAAGGA-3 (Hind III)

P2 5-CCTGGATCCAGCATGATAGATGCCTCCTCTCAA-3 174–3675-CCCAAGCTTCTACCAACCACACTCAATGAAGGA-3

P3 5-TTTGGATCCAGCATGGAGGTACGTGTGC-3 1–1815-CCCAAGCTTCTATTTGAGAGGAGGCATCTATCAT-3

P4 5-AAAGGATCCCAGCAATTGTCACGAGTGGAC-3 77–1815-CCCAAGCTTCTATTTGAGAGGAGGCATCTATCAT-3

P5 5-TTTGGATCCAGCATGGAGGTACGTGTGC-3 1–835-CCCAAGCTTTCAGTCCACTCGTGACAATTGCTG-3

P6 5-TTTGGATCCATGGAGGTACGTGTGC-3 1–445-GGTCTCGAGCTACTTAATCACGTCAGGATCGTG-3(Xho I)

P7 5-TTTGGATCCCACGATCCTGACGTGATTAAG-3 38–645-GGTCTCGAGCTAAGGATTGTCCACTCGTGACAATTGCTG-3

P8 5-TTTGGATCCCACGATCCTGACGTGATTAAG-3 38–445-GGTCTCGAGCTATGGAGGCGCGCCATAATATAG-3

P9 5-TTTGGATCCCTATATTATGGCGCGCCTCCAAC-3 58–855-GGTCTCGAGAGGATTGTCCACTCGTGACAATTGCTG-3

P10 5-TTTGGATCCATGGAGGTACGTGTGC-3 1–225-GGTCTCGAGCTAAGCCCGAATGTAACTGGATGTGATTCC-3

P11 5-TTTGGATCCGGAATCACATCCAGTTACATTCGGGCTC-3 14–445-GGTCTCGAGCTTAATCACGTCAGGATCGTG-3

P12 5-GATCCACGGATGGTGTTTGCTTCCCGCACCATAAGTAGTAAC-3 65–745-TCGAGTTACTACTTATGGTGCGGGAAGCAAACACCATCCGTG-3

P13 5-GATCCTTCCCGCACCATAAGTGCCATCAGCAATTGTAGTAAC-3 70–795-TCGAGTTACTACAATTGCTGATGGCACTTATGGTGCGGGAAG-3

P14 5-GATCCTGCCATCAGCAATTGTCACGAGTGGACAATCCTTAGTAAC-3 75–855-TCGAGTTACTAAGGATTGTCCACTCGTGACAATTGCTGATGGCAG-3

P15 5-GATCCCCTGCGTGCTGGGATTCTAGATCGGCGTGGGATTAGTAAC-3 23–335-TCGAGTTACTAATCCCACGCCGATCTAGAATCCCAGCACGCAGGG-3

P16 5-GATCCAGATCGGCGTGGGATAGTGATATCTTTCACGATTAGTAAC-3 29–395-TCGAGTTACTAATCGTGAAAGATATCACTATCCCACGCCGATCTG-3

P17 5-GATCCAGTGATATCTTTCACGATCCTGACGTGATTAAGTAGTAAC-3 34–445-TCGAGTTACTACTTAATCACGTCAGGATCGTGAAAGATATCACTG-3

P18 5-GATCCATCACATCCAGTTACATTCGGGCTCCTGCGTGCTGGGATTCTTAGTAAC-3 15–295-TCGAGTTACTAAGAATCCCAGCACGCAGGAGCCCGAATGTAACTGGATGTGATG-3

P19 5-GATCCATCACATCCAGTTACATTCGGGCTCCTGCGTGCTGGTAGTAAC-3 15TGTA

TTAGGAAT

(�cpsw(boP(wtPPs2i2co

3i

ap

5-TCGAGTTACTACCAGCACGCAGGAGCCCGAAP20 5-GATCCTACATTCGGGCTCCTGCGTGCTGGGA

5-TCGAGTTACTAATCCCAGCACGCAGGAGCCC

77–181 aa) (Fig. 1A). This result suggested that the 1–83 aa of DRVB might be the dominant antigen region of �B. To define the pre-ise epitopes of �B, the 1–85 aa region was further divided into 15artially overlapping fragments, respectively. The screening resultshowed that P7 (38–64 aa), P9 (58–85 aa), and P12 (65–74 aa),ere recognized by MAb 2F7 (Fig. 1B), while P6 (1–44 aa), P11

14–44 aa), P18 (15–29 aa), and P20 (19–27 aa) were recognizedy 1E5, 4E3, and 5D8 (Fig. 2, a representative image). The resultsf Western blotting showed that P2 (174–367 aa), P4 (77–181 aa),8 (38–44 aa), P10 (1–22 aa), P13 (70–79 aa), P14 (75–85 aa), P1523–33 aa), P16 (29–39 aa), P17 (34–44 aa), and P19 (15–26 aa)ere not recognized by any MAbs (Fig. 1 and Fig. 2, representa-

ive images). Furthermore, MAbs 1E5, 4E3, and 5D8 reacted with20 (19–27 aa) but did not react with the neighboring fragment19 (15–26 aa) and P16 (29–39 aa) (Fig. 2, representative image),o MAbs 1E5, 4E3, and 5D8 only recognized P20 (19–27 aa). MAbF7 recognized P12 (65–74 aa) but did not recognize the neighbor-

ng fragment P13 (70–79 aa), P14 (75–85 aa), and P7 (38–64 aa), soF7 recognize the domain of 65–74 aa. The results of ELISA wereoincident with results of Western blotting (Fig. 3). Two epitopesf DRV �B, P20 (19–27 aa) and P12 (65–74 aa), were identified.

.2. Epitope peptides have good reactogenicity andmmunogenicity

ELISA analysis indicated that both the epitope peptides, P12nd P20, were recognized by DRV positive duck sera and DRV �Bositive mice sera (data not shown). Western blotting and ELISA

–26ACTGGATGTGATG-3TAAC-3 19

–27GTAG-3

showed that mice anti p12 and P20 peptides sera reacted with the�B protein and corresponding epitope peptides, but could not reactwith negative control (sera from uninfected healthy mice).

3.3. Immunofluorescence assay

The IFA experiment showed that only mice anti epitope B seraor MAb 2F7 could react with CEF infected by ARV, but mice antiepitope A sera or MAb 1E5 could not recognize (Fig. 4).

3.4. Homology analysis of the epitopes

Sequence analysis of 12 strains DRV and ARV revealed that theepitope B sequence 19YIRAPACWD27 was completely conserved.Three amino acids of in the epitope A were different between DRVand ARV strains. However, epitopes A and B sequences were com-pletely conserved among DRVs (S12, 89330, and 89026) (Fig. 5).

3.5. Detection of ARV �B antigens

MAbs 1E5 and 2F7, representing recognizing epitopes A and B,respectively, were selected for testing their cross-reactivity withthree heterologous ARV S1133, 99G, and DRV S14 strains in theELISA. The relative binding to heterologous viruses is expressed as

percentage by taking the A405 obtained with DRV S12 in the reac-tion as 100. The results indicated that �B, in cell extracts preparedfrom CEF infected with heterologous ARV S1133, 99G, and DRVS14 strains, was captured by mice anti-�B antiserum (as positive

Y. Li et al. / Virus Research 163 (2012) 546– 551 549

F ane Mi D8, o

coaeC

Fi

ig. 1. SDS-PAGE (up panel) and Western blotting (low panel) analysis (A and B). Lnduced recombinant cells; Lanes 1–5, P1–P5 peptides detected by MAb (1E5/4E3/5

ontrol, PC) (Fig. 6). The relative binding of MAb 2F7 to heterol-

gous virus strains was found to be more than 75%. There are noppreciable differences in the binding of the MAb 2F7 tested. Asxpected, negative results were obtained using the mock infectedEF (<25%) (data not shown). Therefore, MAb 2F7 strongly recog-

ig. 2. SDS-PAGE (left panel) and Western blotting analysis of P6–P20 (right panel). Lane Mnduced recombinant cells; Lanes 6–20, detected by MAb (1E5/4E3/5D8).

, protein molecular marker; Lane C, Protein extracts from the pET30a or pGEX6p-1r 2F7) (A); Lanes 6–14, P1–P14 peptides detected by MAb2F7 (B).

nized ARV S1133, 99G, and DRV S14, suggesting that epitope B is

commonly present among DRV and ARV strains. The relative bind-ing of MAb 1E5 to ARV strains was found to be less than 25%, andbe more than 75% to DRV strains, suggesting that epitope A is onlyexisted in DRV strains (Fig. 6).

, protein molecular marker; Lane C, Protein extracts from the pET30a or pGEX6p-1

550 Y. Li et al. / Virus Research 163 (2012) 546– 551

FMc

4

m

Ffl

ig. 3. Identification of the epitopes by ELISA. Peptides P7, P9, and P12 reacted withAb2F7 (A); peptides P6, P11, and P20 reacted with MAb (1E5/4E3/5D8) (B). No

ross-reactivity was found with pET30a or pGEX6P-1 control (P0).

. Discussion

Regarding the epitope study of reovirus, much effort has beenade with other sigma-class proteins. However, the epitopes of

ig. 4. The �B protein detection by IFA on DEF cells infected with S1133 (400×). Deteuorescencewas found on normal cells detected with MAb 1E5 or mice anti epitope A ser

S12:MEVRVPN FHSFVEG ITSSYIRT PACWDSRS AWDSDIF HDPDVI

89330:----- ------- ----A---- ------- --------- YE--

89026:----- ------- ----A---- ------- --------- YE--

S1133:----- ------- --------- ------A Q-----VT- -V--

OS161:----- ------- --------- ------A Q-----VT- -V--

R2-TW:----- ------- --------- ------A Q-----VT- -V--

2408: --------- ------- --------- --AQ--- --VT--V--

918:------ ------- --------- -----AQ -----VT-- V--

1733: --------- ------- --------- --AQ--- --VT--V--

1017: --------- ------- --------- --AQ--- --VT--V--

Fig. 5. Sequence comparison of epitope A and B on �B of DRV and ARV strains. D

Fig. 6. Cross-reactivity of MAbs 1E5, 2F7, or mice anti DRV S12 sera (PC) to heterol-ogous DRV and ARV strains.

�B have never been identified. Four MAbs 1E5, 4E3, 5D8, and 2F7against �B were developed and could react with recombinant �Band CEF infected with DRV, which demonstrated good specificity(Liu et al., 2010). To locate the epitopes of �B, a set of 20 partiallyoverlapping or consecutive peptides (P1–P20) spanning �B wereexpressed. Using the four MAbs, 1E5, 4E3, 5D8, and 2F7, the pep-tides of P1–P20 were screened by pepscan analysis. Finally, twoantigenic epitopes, 19–27 aa and 65–74 aa, of DRV �B, were iden-tified by Western blotting and ELISA. The MAbs 1E5, 4E3, and 5D8reacted with P20 (19–27 aa) but did not react with the neighboring

fragment P19 (15–26 aa), which has 8 aa overlapping with P20, sug-gesting amino acid 27D is necessary for this epitope. The MAb 2F7reacted with P12 (65–74 aa) but did not react with the neighboringfragments P7 (38–64 aa) and P13 (70–79 aa).

ction with mice anti epitope B sera (A); detection with MAb 2F7 (B); no speciala (C).

KVG GAYCCTQ CCGVLYYGA PPTDGV CFPHHKCHQ77

--- --------- ------- --------- ------- -

--- --------- ------- --------- ------- -

--- --N------ ------- -TL-A--NY ------- -

--- --N------ ------- -TL-A--NY ------- -

--- --N------ ------- -TL----NY ------- -

-----N- --------- ----TL- A--NY---- ----

---- -N------- ------- TL-P--NY- -------

-----N- --------- ----TL- A--NY---- ----

-----N- --------- ----TL- ---NY---- ----

RV S12 sequences were shown on top and thedifferences were indicated.

earch 1

migDpatAbrsdfm

tm(2tca5h1esnMatiot

A

tAa

R

D

Y. Li et al. / Virus Res

Peptides P12 and P20 could be recognized by duck anti DRV orice anti �B sera, suggesting the epitopes have good reactogenic-

ty. After immunization twice with P12 and P20, BALB/c mice couldenerate the corresponding antibody, which could be detected byRV, �B and the epitope peptides, suggesting the epitope A and Beptides have good immunogenicity. Compared with various DRVnd ARV strains, the peptides P20 is totally homologous. The reac-ion to DRV and ARV by IFA and cross-reactivity of MAbs to DRV andRV strains by ELISA also confirmed that P20 is commonly sharedy DRV and ARV strains. Mice anti P12 sera and MAb 1E5 did notecognize ARV by IFA and ELISA suggested that epitope A is DRV-pecific epitope. The MAb 2F7 might be suitable candidates for theiagnosis of DRV and ARV isolates, while MAb 1E5 could be usedor discriminating ARV or DRV infection. Conservation of epitope B

ay be useful in view of the biological function of �B.The alignments of �B deduced amino acids sequences DRV, ARV,

urkey reovirus (TRV), and Nelson Bey virus (NBV) revealed thatost conserved amino acids occupied at the N-terminal region

1–113 aa) (Zhang et al., 2007a,b; Day et al., 2007; Sellers et al.,004; Kapczynski et al., 2002). The results that two epitopes iden-ified by four MAbs both located at N-terminal region of �B areonsistent with these recent investigations. MRV �3 (Mabrouknd Lemay, 1994; Schiff et al., 1988), a similar zinc-binding motif1C–X2–C–X17–H–X2–C75 within the DRV �B protein sequence,as been described (Zhang et al., 2007b; Le Gall-Recule et al.,999), which is important for proper folding and stability (Mabroukt al., 1994; Shepard et al., 1996). The report that the criticalequence for MRV �3 interaction with �1 lie in the amino termi-us of the molecule (Liemann et al., 2002; Shepard et al., 1996).utations in the zinc-binding motif of �3 eliminate its ability to

ssociate with capsid protein �1 (Shepard et al., 1996). The epi-ope A 65TDGVCFPHHK74 located in zinc finger motif, suggestingt should be functional in �B folding and interaction with �A (anal-gous of �1) protein. Further experiments are necessary to confirmhese functional roles.

cknowledgements

This work was supported by a grant from the China Agricul-ure Research System (CARS-43-10) and Chinese Special Fund forgro-scientific Research in the Public Interest (201003012). Welso thank Dr. SungMo Son for giving proofread for this manuscript.

eferences

ay, J.M., Pantin-Jackwood, M.J., Spackman, E., 2007. Sequence and phylogeneticanalysis of the S1 genome segment of turkey-origin reoviruses. Virus Genes 35,235–242.

63 (2012) 546– 551 551

Heffles-Redmann, U., Muller, H., Kaleta, E.F., 1992. Structural and biological char-acteristics of reoviruses isolated from Muscovy ducks (Cairina moschata). AvianPathol. 21, 481–491.

Hou, H.S., Su, Y.P., Shieh, H.K., Lee, L.H., 2001. Monoclonal antibodies against differentepitopes of nonstructural protein �NS of avian reovirus S1133. Virology 282,168–175.

Huang, P.H., Ying, J., Li, Y.J., Su, Y.P., Lee, L.H., Liu, H.J., 2005. Epitope mapping andfunctional analysis of sigma A and sigma NS proteins of avian reovirus. Virology332, 584–595.

Kapczynski, D.R., Sellers, H.S., Simmons, V., Schultz-Cherry, S., 2002. Sequence anal-ysis of the S3 gene from a turkey reovirus. Virus Genes 25, 95–100.

Kaschula, V.R., 1950. A new virus disease of the Muscovy duck (Cairina moschataLinn.) present in Natal. J. S. Afr. Vet. Med. Assoc. 21, 18–26.

Kuntz-Simon, G., Le Gall-Recule, G., de Boisseson, C., Jestin, V., 2002. Muscovy duckreovirus �C protein is a typically encoded by the smallest genome segment. J.Gen. Virol. 83, 1189–1200.

Le Gall-Recule, G., Cherbonnel, M., Arnauld, C., Blanchard, P., Jestin, A., Jestin, V.,1999. Molecular characterization and expression of the S3 gene of Muscovy duckreovirus strain 89026. J. Gen. Virol. 80, 195–203.

Liemann, S., Chandran, K., Baker, T.S., Nibert, M.L., Harrison, S.C., 2002. Structure ofthe reovirus membrane-penetration protein, �1, in a complex with its protectorprotein, �3. Cell 108, 283–295.

Liu, M., Chen, X., Wang, Y., Zhang, Y., Li, Y., Wang, Y., Shen, N., Chen, H., 2010. Charac-terization of monoclonal antibodies against Muscovy duck reovirus �B protein.Virol. J. 7, 133.

Mabrouk, T., Lemay, G., 1994. Mutations in a CCHC zinc-binding motif of thereovirus sigma 3 protein decrease its intracellular stability. J. Virol. 68,5287–5290.

Malkinson, M., Perk, K., Weisman, Y., 1981. Reovirus infection of young Muscovyducks (Cairina moschata). Avian Pathol. 10, 433–440.

McNulty, M.S., 1993. Reovirus. In: McFerran, J.B., McNulty, M.S. (Eds.), Virus Infec-tions of Birds. Elsevier Science Publishers, Netherlands, pp. 181–193.

Robertson, M.D., Wilcox, G.E., 1986. Avian reovirus. Vet. Bull. 56, 154–174.Rosenberger, J.K., Olson, N.O., 1991. Reovirus infections. In: Calnek, B.W., Barnes, H.J.,

Beard, C.W., Reid, W.M., Yoder, H.W. (Eds.), Diseases of Poultry. , 9th ed. IowaState University Press, Ames, IA, pp. 639–647.

Schiff, L.A., Nibert, M.L., Co, M.S., Brown, E.G., Fields, B.N., 1988. Distinct binding sitesfor zinc and double-stranded RNA in the reovirus outer capsid protein �3. Mol.Cell. Biol. 8, 273–283.

Sellers, H.S., Linnemann, E.G., Lorena, P., Kapczynski, D.R., 2004. Phylogeneticanalysis of the sigma 2 protein gene of turkey reoviruses. Avian Dis. 48,651–657.

Shepard, D.A., Ehnstrom, J.G., Skinner, P.J., Schiff, L.A., 1996. Mutations in the zinc-binding motif of the reovirus capsid protein �3 eliminate its ability to associatewith capsid protein �1. J. Virol. 70, 2065–2068.

Vakharia, V.N., Edwards, G.H., Annadata, M., Simpson, L.H., Mundt, E., 1996. Cloning,sequencing and expression of the S1 and S3 genome segments of avian reovirusstrain 1733. In: Proceedings of the International Symposium on AdenovirusReovirus Infections in Poultry Rauschhorzhausen, Germany, pp. 168–180.

Wichramasinghe, R., Meanger, J., Enriguez, C.E., Wilcox, G.E., 1993. Avian reovirusproteins associated with neutralization of virus infectivity. Virology 194,688–696.

Zhang, Y., Guo, D.C., Geng, H.W., Liu, M., Hu, Q.L., Wang, J.W., Tong, G.Z., Kong,X.G., Liu, N.H., Liu, C.G., 2007a. Characterization of M-class genome segmentsof Muscovy duck reovirus S14. Virus Res. 125, 42–53.

Zhang, Y., Guo, D.C., Liu, M., Geng, H.W., Hu, Q.L., Liu, Y., Liu, N.H., 2007b. Character-ization of the �B-encoding genes of Muscovy duck reovirus: �C–�B-ELISA for

antibodies against duck reovirus in ducks. Vet. Microbiol. 121, 231–241.

Zhang, Y., Liu, M., Shuidong, O., Hu, Q.L., Guo, D.C., Chen, H.Y., Han, Z., 2006. Detectionand identification of avian, duck, and goose reoviruses by RT-PCR: goose andduck reoviruses are part of the same genogroup in the genus Orthoreovirus. Arch.Virol. 151, 1525–1538.