Identification of Goose-Origin Parvovirus as a Cause of NewlyEmerging Beak Atrophy and Dwarfism Syndrome in Ducklings

Kexiang Yua Xiuli Maa Zizhang Shengb Lihong Qia Cunxia Liua Dan Wangc Bing Huanga Feng Liacd Minxun Songa

Institute of Poultry Science Shandong Academy of Agricultural Sciences Jinan Shandong Chinaa Department of Biochemistry and Molecular Biophysics ColumbiaUniversity New York New York USAb Department of Biology and Microbiology South Dakota State University Brookings South Dakota USAc Department of Veterinaryand Biomedical Sciences South Dakota State University Brookings South Dakota USAd

A recent epizootic outbreak in China of duck beak atrophy and dwarfism syndrome (BADS) was investigated using electronmicroscopic genetic and virological studies which identified a parvovirus with a greater similarity to goose parvovirus (GPV)(97 protein homology) than to Muscovy duck parvovirus (MDPV) (90 protein homology) The new virus provisionally des-ignated GPV-QH15 was found to be antigenically more closely related to GPV than to MDPV in a virus neutralization assayThese findings were further supported by phylogenetic analysis showing that GPV-QH15 evolved from goose lineage parvovi-ruses rather than from Muscovy duck- or other duck species-related parvoviruses In all two genetic lineages (GPV I and GPVII) were identified from the GPV samples analyzed and GPV-QH15 was found to be closely clustered with two known goose-origin parvoviruses (GPVa2006 and GPV1995) together forming a distinctive GPV IIa sublineage Finally structural modelingrevealed that GPV-QH15 and the closely related viruses GPVa2006 and GPV1995 possessed identical clusters of receptor-inter-acting amino acid residues in the VP2 protein a major determinant of viral receptor binding and host specificity Significantlythese three viruses differed from MDPVs and other GPVs at these positions Taken together these results suggest that GPV-QH15 represents a new variant of goose-origin parvovirus that currently circulates in ducklings and causes BADS a syndromereported previously in Europe This new finding highlights the need for future surveillance of GPV-QH15 in poultry in order togain a better understanding of both the evolution and the biology of this emerging parvovirus

The Parvoviridae are a family of small DNA viruses that consistsof two subfamilies Parvovirinae and Densovirinae Parvovi-

ruses are members of the subfamily Parvovirinae and can infectand cause diseases in humans as well as in other mammals water-fowl and poultry (1) In waterfowl two genetically related parvo-virus subgroups goose parvovirus (GPV) and Muscovy duck par-vovirus (MDPV) have been identified (2) GPV primarily infectsgeese and Muscovy ducks and can be highly contagious with 70to 100 mortality in goslings under the age of 4 weeks (3 4)Interestingly MDPV differs from GPV in that it infects andcauses disease in Muscovy ducks and several hybrid duck breedsonly not geese Both GPV and MDPV have been assigned phylo-genetically to the genus Dependovirus in the family Parvoviridaedue to similar genetic properties and evolutionary origins Thetwo viruses possess approximately 85 homology in their overallprotein sequences and have been shown to be antigenically relatedto each other Nonetheless GPV and MDPV are distinct and as aresult any antibody-driven cross-protection between these twoclosely related viruses is largely limited (5 6)

Clinically GPV and MDPV are similar in that the two virusesare excreted in large amounts in the feces of infected waterfowlsubsequently spreading rapidly to susceptible birds by both directand indirect routes and resulting in large fatal outbreaks on re-gional duck and goose farms (7 8) The disease initially manifestswith watery diarrhea wheezing and locomotor dysfunction fol-lowed by sudden death Young waterfowl that survive an acuteinfection often show profound growth retardation along with lossof feathers and reddening of the skin particularly on the backInfection and mortality rates are much lower in adult ducks andgeese but affected birds show growth retardation and reducedproduction performance postinfection

Here we report on the isolation and characterization of a novel

parvovirus from ducks displaying clinical signs and symptomsGenetic and antigenic analysis showed that the new virus is moreclosely related to GPV than to MDPV Clinically instead of show-ing symptoms normally associated with GPV or MDPV infectionstypically seen in ducks and geese ducks infected with this novelparvovirus provisionally designated GPV-QH15 developed amore severe syndrome beak atrophy and dwarfism syndrome(BADS)

MATERIALS AND METHODSEthics statement Invasive procedures primarily the inoculation of virusinto eggs by an allantoic route were used in this study These eggs wereharvested by day 15 from the first day of egg hatching and all live chickenembryos were euthanized by placing them at 4degC overnight These proce-dures were discussed and approved by the Institutional Animal Care andUse Committee of the Shandong Poultry Institute (approval number 12-018) All samples for viral genome detection and virus isolation werecollected from ducklings on infected poultry farms located in Shandongand other provinces in the major poultry production region of China

Received 10 December 2015 Returned for modification 30 December 2015Accepted 12 May 2016

Accepted manuscript posted online 18 May 2016

Citation Yu K Ma X Sheng Z Qi L Liu C Wang D Huang B Li F Song M 2016Identification of goose-origin parvovirus as a cause of newly emerging beakatrophy and dwarfism syndrome in ducklings J Clin Microbiol 541999 ndash2007doi101128JCM03244-15

Editor B W Fenwick University of Tennessee Knoxville

Address correspondence to Feng Li fenglisdstateedu orMinxun Song mxsongaliyuncom

Copyright copy 2016 American Society for Microbiology All Rights Reserved

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Local government institutions approved this work None of these sampleswere collected from farms with endangered or protected species

Virus isolation Spleen and liver tissues from diseased ducklings werecollected and were homogenized in sterile phosphate-buffered saline(PBS pH 72) to form a 20 (wtvol) suspension containing penicillinand streptomycin After centrifugation at 1106 g for 20 min the super-natants were filtered through 02-m-pore-size syringe-driven filters Thefiltered suspensions were then inoculated into 9-day-old specific-patho-gen-free (SPF) chicken embryonic eggs 10-day-old SPF duck or meatduck embryonic eggs 10-day-old SPF mallard duck embryonic eggs and12-day-old SPF chicken embryonic eggs at a dose of 01 ml per egg Em-bryonic eggs were examined daily and the allantoic fluids of dead embry-onic eggs were harvested 2 to 6 days after inoculation These were furtherpassaged blindly three times in the corresponding SPF eggs The allantoicfluids derived from each passage were then harvested and were stored at80degC for further testing as described below

HAs To determine whether the allantoic fluids collected from variousSPF eggs were capable of agglutinating red blood cells hemagglutinationassays (HAs) were performed using red blood cells from several avianspecies including chickens ducks geese and pigeons as well as frommice and rabbits

Electron microscopy Negative-contrast scanning electron micros-copy was performed to detect viral particles The harvested allantoic fluidsfrom dead SPF goose embryonic eggs were placed in 1 ml of double-distilled water and were centrifuged at 1106 g for 20 min The super-natant was placed on Formvar-coated copper grids (Electron MicroscopyServices) and was stained with 2 phosphotungstic acid for 2 min Scan-ning electron microscopy was performed to detect viral particles presentin the samples All electron microscopy work was conducted at Jilin Uni-versityrsquos electron microscopy core facility

PCR and RT-PCR Viral RNA was extracted from the allantoic fluidswith the MiniBEST viral RNA extraction kit (TaKaRa) according to the

manufacturerrsquos instructions and was screened by a viral reverse transcrip-tion-PCR (RT-PCR) panel targeting viral diseases of ducklings caused byRNA viruses including duck hepatitis virus (DHV) mallard duck reovi-rus (MDRV) newly emerged duck reovirus variant (NDRV) and duckTembusu virus (DTMUV) Concurrently viral DNA was extracted withDNAiso reagent (TaKaRa) and was analyzed using a viral PCR panel tar-geting duckling DNA viruses in particular duck enteritis virus (DEV)Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV)

Full-genome sequencing and analysis Following confirmation thatan embryonic egg-derived fluid was positive for goose parvovirus thevirus was purified by ultracentrifugation and the viral genome was ex-tracted Four pairs of primers were designed and synthesized according tothe GPV genome sequence information deposited in GenBank (Table 1)These primers amplified four overlapping regions that encompassed thecomplete genome of this new parvovirus PCRs were performed usingthese primers which resulted in four overlapping fragments making upthe full length of the viral genome The Sanger-based sequencing methodwas used to determine the sequence of the amplified fragment and denovo genome assembly was employed to assemble the full viral genome

The 5= end of the genome was amplified using 5=-Full RACE kits(TaKaRa) and all PCR products were purified and were cloned intopMD18-T vectors (TaKaRa) for sequencing Goose parvovirus was usedas the template for the assembly of the contigs using the Lasergene pro-gram (DNAStar)

Phylogenetic tree analysis on the full viral genome included NS1 VP1and VP2 sequences and was performed using PhyML (9) The TN93Gmodel was evaluated and was determined to be one of the best substitu-tion models by use of MEGA6 (10) The approximate likelihood ratio test(aLRT) method was used to estimate the statistical support of branchingpatterns The tree was rooted using the branch between MDPVs andGPVs

TABLE 1 Summary of virus strains used in this study

Strain Yr Host Location GenBank accession noName used inthis study

Source orreference

QH15 2015 Peking duck Shandong China KT751090 GPV-QH15 This study82-0321V 2006 Goose Taiwan EU583389 GPVa2006 6YZ99-6 1999 Goose Jiangsu China KC996730 GPVa1999 GenBanka

LH 2012 Goose China KM272560 GPVa2012 GenBankGDaGPV 1978 Goose Guangdong China HQ891825 GPV1978 GenBankYZ 2013 Goose Anhui China KR091960 GPVa2013 GenBankWX 2013 Goose Anhui China KR091959 GPVb2013 GenBankE 2012 Goose Anhui China KC184133 GPVb2012 GenBank06-0329 2006 Goose Taiwan EU583391 GPVb2006 6Yan-2 2013 Yan goose Anhui China KR136258 GPVc2013 GenBankY 2011 Muscovy duck Anhui China KC178571 GPV2011 GenBankSH 2009 Anser anser Shanghai China JF333590 GPV2009 GenBankVirulent B 1995 Anser anser Hungary U25749 GPV1995 2MDPV-GX5 2011 Muscovy duck Guangxi China KM093740 MDPV2011 23SHFX1201 2012 Swan Shanghai China KC478066 GPVc2012 GenBankSAAS-SHNH 2012 Muscovy duck Shanghai China KC171936 MDPVb2012 GenBankFM 1993 Cairina moschata Hungary NC_006147 MDPV1993 2P 1988 Muscovy duck Fujian China JF926697 MDPV1988 GenBankHBZF07 2007 Goose Hebei China EU022755 GPVa2007 GenBankH1 2001 Goose Heilongjiang China JQ409356 GPVa2001 GenBankHG582 1982 Goose Heilongjiang China AY506546 GPV1982 GenBankLN-106 2006 Goose Liaoning China EU253479 GPVc2006 GenBank01-1001 2001 Goose Taiwan AY382889 GPVb2001 799-0808 1999 Goose Taiwan AY382888 GPVb1999 7GDFSh 2007 Goose Guangdong China EU088103 GPVb2007 GenBankSCh 2007 Goose Sichuan China EU088101 GPVc2007 GenBankGD 2003 Muscovy duck Guangdong China AY510603 MDPV2003 GenBanka Information available at ncbinlmnihgovGenBank

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Virus neutralization and antigenic relationship To determine theantigenic similarity between the newly isolated parvovirus and the twoother known waterfowl parvoviruses (GPV and MDPV) reciprocal virusneutralization was performed with these three viruses and their corre-sponding antisera The representative GPV and MDPV strains selected forthis work were GY13 and AH11 respectively Briefly the heat-inactivatedantisera 5-fold serially diluted were each mixed with 200 50 embryo-lethal doses (ELD50) of each of the three viruses After the mixture wasincubated at 37degC for 1 h 10-day-old SPF duck embryonic eggs wereinoculated via the allantoic route Each dilution was tested in five em-bryos and the infected eggs were checked daily Dead embryos were re-corded for as long as 6 days postinoculation The viral neutralization titerwas determined by taking the reciprocal of the log2 of the highest dilutionthat inhibited 50 of embryo death Antibody titers were calculated withthe formula of Reed and Muench The titers presented below are the meanvalues for three repeated experiments The antigenic relationship was de-termined by the equation r1 middot r2 where R is the antigenic relatednessindex r1 is the ratio of the neutralizing antibody titers of virus ldquoBrdquo-specificantiserum against the heterologous virus ldquoArdquo to the neutralizing antibodytiters of virus ldquoArdquo-specific antiserum against the homologous virus ldquoArdquoand r2 is the ratio of the neutralizing antibody titers of virus ldquoArdquo-specificantiserum against the heterologous virus ldquoBrdquo to the neutralizing antibodytiters of virus ldquoBrdquo-specific antiserum against the homologous virus ldquoBrdquoThe lower the R value the greater the antigenic divergence between twoviruses

Structural modeling of the viral VP2 protein HHpred was used tosearch for the optimal PDB structures for building a homolog model ofthe VP2 of GPV-QH15 PDB 3NG9 (httpwwwrcsborg) the resolvedcapsid structure of a serotype I adeno-associated virus (AAV) was chosenas the template and was aligned to the VP2 of GPV-QH15 Modeller (11)was used to build the structure model which was checked using Procheck(12ndash14) The viral surface was then generated using PyMOL with thesymmetric information from 3NG9

Screening of clinical samples for the presence of the novel parvovi-rus by PCR The 160 samples used consisted of 120 fecal swabs and 40spleen or liver tissue samples from ducklings retrieved from outbreakpoultry farms originating from five provinces of China Shandong (n 90) Jiangsu (n 30) Hebei (n 20) Anhui (n 10) and Jiangxi (n 10) Additionally 100 fecal samples were collected from healthy ducklingsraised under similar conditions in the same region from which the 160infected samples were derived for use as controls All samples were ana-lyzed for the novel parvovirus using PCR targeting the conserved capsidVP3 gene The sequence of the forward primer was 5= TGTTCGCTCATTCACAGGA 3= and the sequence of the reverse primer was 5= TATGGTTTCCACCCTACGC 3= the length of the amplified fragment was 454 bp

Nucleotide sequence accession number The genome sequence of thenew parvovirus determined in this study was submitted to GenBank un-der accession no KT751090 (httpwwwncbinlmnihgovnuccoreKT751090)

RESULTSClinical symptoms In October 2014 several cases of a new dis-ease in commercial meat ducks were reported in Jiangsu provinceChina The outbreaks have since spread to several primary duckproduction provinces including Shandong Hebei Henan An-hui and Jiangxi (Fig 1) The cases were typically characterizedclinically by tongue protrusion accompanied by a shortened beakwith approximately 10 to 30 morbidity and 2 to 6 mortality atthe end of the growth period (Fig 2A) The final beak size wasapproximately 10 to 30 that of the normal beak of a healthyduckling Poor beak development resulted in protruded tonguesin previously infected ducklings significantly affecting their abil-ity to eat and drink (Fig 2A and B) As a result the diseasedducklings continued to have poor growth and increased mortality

In most cases the affected ducklings had fragile and easily brokenlegs potentially caused by the progressive loss of skeletal musclemass in the legs Initial symptoms including the shortened beakand tongue protrusion began at the age of 6 days and peaked at 3weeks Infection rates were much lower in ducks above the age of3 weeks and symptoms likely became subclinical All major duckspecies in this study were found to be susceptible to this new dis-ease and both males and females were represented among thecases The morbidity rate was variable among affected duck flocksranging from 10 to 30 Although the 2-to-6 mortality rateobserved for affected farms was not high the production perfor-mance of ducks that survived the infection was significantly re-duced due to abnormal feathering and growth retardation

Isolation of a novel parvovirus from ducklings with BADSIn June 2015 liver and spleen tissue samples from ducklings ex-hibiting BADS were submitted to the Shandong Poultry ResearchInstitute in Jinan China for virus isolation and diagnosis Theinitial pathogen screening on these samples by real-time reversetranscription-PCR (rt-RT-PCR) or PCR assays was positive onlyfor goose parvovirus tests for other common RNA or DNA vi-ruses of waterfowl were negative In the inoculated SPF embryoniceggs from geese and Muscovy ducks embryo deaths occurred onthe second passage and parvovirus particles with a diameter of 20to 22 nm were detected in the allantoic fluids by scanning electronmicroscopy (Fig 2C) Like GPV the isolated virus could not ag-glutinate red blood cells from tested animal species These datasuggested that the isolated virus is a member of the parvoviruses Ithas been provisionally designated strain GPV-QH15

Genome sequencing and analysis Sequence analysis showedthat the complete genome of GPV-QH15 was 5048 bp long andcontained two major open reading frames (ORFs) related to thoseof other parvoviruses isolated from waterfowl The ORF locatedon the left side of the viral genome encodes the 627-amino-acid(627-aa) nonstructural (NS) polyprotein which is cleaved intothe NS1 (627 aa) and NS2 (451 aa) proteins These two proteins

FIG 1 Geographical distribution of reported BADS outbreaks in China Prov-inces where BADS cases have been confirmed since October 2014 are shown inred These provinces include Shandong Jiangsu Henan Hebei Beijing Zhe-jiang Jiangxi Anhui Hubei Shanghai Fujian Guangdong and GuangxiNote that no disease outbreak was reported in provinces shown in blue yellowor green

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share the same termination code during protein translation butpossess different start codons The ORF on the right side of theviral genome encodes the capsid polyprotein of 732 aa which isfurther divided into VP1 (732 aa) VP2 (587 aa) and VP3 (534 aa)The three capsid proteins share a C terminus and stop codon butdiffer at the N-terminal region The viral genome also contains the415-bp inverted terminal repeats (ITRs) at both ends of the DNAIn a protein database search GPV-QH15 showed greater similar-ity to goose parvovirus (GPV) (97 protein homology) than toMuscovy duck parvovirus (MDPV) (90 protein homology) Itshould be noted as well that GPV-QH15 is very similar to a duck-origin GPV-related parvovirus isolated from Cherry Valley duck-lings (15 16) termed SDLC01 these two viruses share approxi-mately 993 homology in their viral genome sequences Weincluded this viral sequence in our further phylogenetic analysis

A phylogenetic tree was inferred from the full-genome se-quences of our isolated GPV-QH15 DNA and the full genomes ofother waterfowl parvoviruses that had been determined previ-ously (Fig 3A) GPV-QH15 was found to have diverged mostrecently from GPVa2006 and GPV1995 GPVa2006 was a goose-origin parvovirus and could produce 100 mortality when inoc-ulated into 1-day-old Muscovy ducklings (6) Interestingly GPV1995was a goose parvovirus discovered in Hungary (isolated from dis-eased ducklings in 1995) that had also been thought to be associ-ated with a short beak syndrome and growth retardation symp-toms similar to the clinical symptoms caused by GPV-QH15 (17)

On the basis of this full-genome phylogenetic analysis it ap-pears that GPV-QH15 is a goose-origin parvovirus All GPVs an-alyzed seem to have evolved into two distinct genetic lineagestermed GPV I and GPV II In GPV II two sublineages GPV IIaand GPV IIb were observed GPV-QH15 and the closely relatedvirus GPVa2006 were two core components in the GPV IIa sub-lineage distinct from the GPV IIb sublineage Phylogenetic treeswere also determined for the nucleotide sequences of the viral NS1(Fig 3B) VP1 (Fig 3C) and VP2 (Fig 3D) genes Although thephylogenetic assignment of several viruses to each genetic lineageor each sublineage was variable all the trees had comparable to-pologies The most considerable difference is in the gene tree forviral NS1 (Fig 3B) which did not group GPV1995 together withGPV-QH15 and GPVa2006 not even placing GPV1995 in theGPV IIa sublineage Meanwhile the gene trees derived from theviral VP1 and VP2 genes placed GPV1995 in the GPV IIa sublin-eage (Fig 3C and D) in accordance with the tree derived from thecomplete-genome analysis (Fig 3A) The close association ofGPV-QH15 with GPV1995 is near-conclusive evidence thatGPV1995 or its ancestor was an original source of GPV-QH15 Inthe VP2 tree we also included for analysis additional 10 GPV-QH15-like strains These strains included SDLC01 reported pre-viously by Diao and colleagues (15) and 9 field strains that wecollected from clinical samples (see Table 4) Again these 2015isolates along with GPV-QH15 clustered together with GPV1995to form a distinctive genetic lineage

FIG 2 Clinical signs in infected ducklings (A and B) and electron micrograph of parvoviruses isolated from diseased ducklings (C) (A) A duck flock infected withGPV-QH15 The red arrow indicates one diseased duckling with BADS (B) Tongue protrusion was present in dead ducklings from a poultry farm with a diseaseoutbreak Red arrows indicate diseased ducklings while the green arrow indicates a healthy duckling (C) Negative staining shows the presence of parvovirusparticles (indicated by the black arrow) Bar 200 nm

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Cross-neutralization between the newly identified parvovi-rus GPV-QH15 and a closely related GPV or MDPV To deter-mine the cross-neutralization and antigenic relationship betweenGPV-QH15 and a closely related GPV or MDPV we performed anembryonic egg-based virus neutralization assay Two representa-tive strains GPV-GY13 and MDPV-AH11 and their correspond-ing antisera were used in this assay As summarized in Table 2 theantigenic relatedness index between GPV-QH15 and GPV-GY13(r 058) was greater than that observed between GPV-QH15

and MDPV-AH11 (r 020) We also noted that the antigenicdistance between circulating GPV-GY13 and MDPV-AH11 (r 005) was greater than that between the newly emerged virus GPV-QH15 and MDPV-AH11 (r 020) These data indicated thatGPV-QH15 despite its prevalence in ducklings is more closelyrelated to GPVs than to MDPVs

Structural modeling of the VP2 protein To provide struc-tural insights into how GPV-QH15 succeeds in establishing pro-ductive and sustained transmission in ducklings we created a

FIG 3 Maximum likelihood trees inferred from the nucleotide sequences of the complete genomes (A) NS1 genes (B) VP1 genes (C) and VP2 genes (D) ofGPV-QH15 and other goose and duck parvoviruses The levels of statistical support (determined by the aLRT method) for the most internal branches are shownThe designations and sources of the virus strains used in this analysis are given in Table 1 The viruses used for analysis (with their taxonomic names given inparentheses) are as follows GPV-QH15 (QH152015DuckChina) GPVa2006 (82-0321V2006GooseTaiwan) GPVa1999 (YZ99-61999GooseChina)GPVa2012 (LH2012GooseChina) GPV1978 (GDaGPV1978GooseChina) GPVa2013 (YZ2013GooseChina) GPVb2013 (WX2013GooseChina)GPVb2012 (E2012GooseChina) GPVb2006 (06-03292006GooseTaiwan) GPVc2013 (Yan-22013GooseChina) GPV2011 (Y2011DuckChina)GPV2009 (SH2009GooseChina) GPV1995 (Virulent B1995GooseHungary) MDPV2011 (MDPV-GX52011DuckChina) GPVc2012 (SHFX12012012SwanChina) MDPVb2012 (SAAS-SHNH2012DuckChina) MDPV1993 (FM1993DuckHungary) MDPV1988 (P1988DuckChina) GPVa2007(HBZF072007GooseChina) GPVa2001 (H12001GooseChina) GPV1982 (HG5821982GooseChina) GPVc2006 (LN-1062006GooseChina)GPVb2001 (01-10012001GooseTaiwan) GPVb1999 (99-08081999GooseTaiwan) GPVb2007 (GDFSh2007GooseChina) GPVc2007 (SCh2007GooseChina) MDPV2003 (GD2003DuckChina) GPV-GX-2015 (GX2015DuckChina) GPV-HB-2015 (HB2015DuckChina) GPV-HN-2015 (HN2015DuckChina) GPV-JS-2015 (JS2015DuckChina) GPV-SDZJ (ZJ2015DuckChina) GPV-SDHZ-2015 (SDHZ2015DuckChina) GPV-SDLC-2015(SDLC2015DuckChina) GPV-SDLY-2015 (SDLY2015DuckChina) GPV-SDLC01-2015 (SDLC012015DuckChina) See Table 1 for more informationabout these viruses

TABLE 2 Virus neutralization index and antigenic relatedness

Virus strain

Antibody titer (log10) of the following antiserum R indexa for the following strain

GPV-GY13 MDPV-AH11 GPV-QH15 GPV-GY13 MDPV-AH11 GPV-QH15

GPV-GY13 292 222 257 1MDPV-AH11 187 280 222 005 1GPV-QH15 210 162 222 058 020 1a The antigenic relatedness index is described in Materials and Methods

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structural model of the viral VP2 protein based on the solvedX-ray crystallographic structure of a human adeno-associated vi-rus (AAV) VP2 protein (httpwwwrcsborg) Numerous studieshave shown that the VP2 protein is a primary driving force incellular receptor recognition and thus host specificity (18) Theoverall sequence identity of 56 in the VP2 protein between AAVGPVs and MDPVs allowed us to predict several important struc-tural features One such feature was the highly conserved recep-tor-binding pocket of the VP2 protein Structural modeling fo-cused on the identification of the variable viral surface-orientatedamino acid residues that are believed to play a role in the host-switching mechanism of GPV-QH15

As summarized in Fig 4 our structural modeling and proteinsequence alignment identified a group of eight positions on theVP2 protein that could distinguish MDPVs from GPVs Theseresidues were chosen because they were highly conserved in onegroup but variable in another group It should be noted thatstrain-specific mutations were not included in our analysis Fourof the eight positions were located on the viral surface (Table 3Fig 4B)While VP2 position 305 is located in the 3-fold symmetriccenter of the viral capsid the other three positions (residues 392430 and 558) are located in the vicinity of the canyon where thevirusndash host receptor interaction occurs Intriguingly these threesurface-exposed residues (residues 392 430 and 558) in GPV-QH15 differed from those in both MDPVs and other GPVs butwere the same as those in the closely related viruses GPV1995 andGPVa2006 Specifically GPV-QH15 GPV1995 and GPVa2006possessed identical patterns of amino acids at these three posi-tions I392 R430 N558 In comparison with GPV-QH15 allMDPVs analyzed evolved into two groups one with I392 L430

N558 and the other with L392 K430 N558 while all other GPVswere completely conserved (L392 K430 D558) at these three po-sitions It is noteworthy that the invariant residue asparagine (N)was present at VP2 position 558 in all MDPVs analyzed GPV-

FIG 4 Structural modeling of the VP2 protein (A) Cartoon view of the monomer of the VP2 protein of GPV-QH15 Mutated positions in the VP2 proteindistinguishable between duck and goose parvoviruses are shown in magenta (B) VP2 mutations located on the viral surface The 3-fold and 5-fold symmetry axesare outlined with a triangle and a pentagon respectively The canyon that connects two 5-fold symmetry centers is the receptor-binding site (outlined with anellipse) (20 21) Four of the eight mutations that are located on the viral surface are shown in magenta The same mutations in symmetric units are connectedusing dashed lines While VP2 position 305 falls in the 3-fold symmetric center positions 392 430 and 558 are close to the canyon (the receptor binding site)

TABLE 3 Amino acid differences at the surface-exposed positions 305392 430 and 558 in the VP2 protein among the newly emergent virusGPV-QH15 the closely related viruses GPV1995 and GPVa2006MDPVs and other GPVs

Virusa

Amino acid position of the VP2 protein

305 392 430 558

GPV-QH15 N I R NMDPV1988 G I L NMDPV1993 G I L NMDPV2003 G I L NMDPV2011 N L K NMDPVb2012 N L K NGPV1978 S L K DGPV1995 S I R NGPVa1999 S L K DGPVa2006 S I R NGPVb2006 S L K DGPV2011 S L K DGPVa2012 S L K DGPVb2012 S L K DGPVc2012 S L K DGPVa2013 S L K DGPVb2013 S L K DGPVc2013 S L K Da These viruses were also used for phylogenetic tree analysis (Fig 3)

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QH15 and GPV1995 while aspartic acid (D) structurally similarto N with a negative charge potential was found at this positionamong all GPVs analyzed In addition the VP2 protein sequencesof GPV-QH15 SDLC01 and nine other field isolates had approx-imately 997 identity All these viruses possessed identical aminoacid residues at the three positions of VP2 as discussed above

Molecular epidemiology To establish the clinical relevance ofthis novel virus in causing BADS in ducklings 120 fecal swabs and40 tissue samples from infected animals with or without BADS aswell as 100 fecal swabs from our uninfected control group werecollected and were analyzed for the presence of the viral VP3 genesequence (Table 4) Our results showed that all 33 tissue samples(100) from diseased ducklings exhibiting tongue protrusiontested positive for the virus and 63 of the 82 fecal swabs (77)were also positive by the PCR test Furthermore we found that 7 of12 total tissue samples (58) and 15 of 38 total fecal swabs (39)from ducklings without any noticeable syndrome but on farmswith disease were positive for the virus None of the 100 controlfecal samples derived from clinically healthy ducklings tested pos-itive as expected This strong presence of GPV-QH15 in clinicalsamples from diseased ducklings indicates that the parvovirus isthe likely etiological cause of BADS in these cases

DISCUSSION

This study was undertaken to investigate the etiological cause of arecent outbreak of BADS in China Parvovirus-associated BADSin ducklings was initially described in Europe by Palya and col-leagues in 2009 (17) Disease outbreaks were discovered predom-inantly in the major poultry-producing provinces of easternChina The results of our studies strongly support a goose-originparvovirus GPV-QH15 as the cause of the BADS prevalent dur-ing this outbreak

Full-genome phylogenetic tree analysis provides the most con-clusive evidence of the genetic evolution and lineage classificationof GPV-QH15 among duck and goose parvoviruses by reducingany bias imparted by using various portions of the genome withmore-variable evolutionary rates While the NS1 genes are themost conserved among individual parvovirus gene sequences theVP1 and VP2 genes are considerably more diverse Accordingly itis reasonable to expect that phylogenetic trees derived from anal-ysis of these three genes may show some differences in their topol-ogies Even in light of this phylogenetic trees inferred from each ofthree individual genes as well as from the complete viral genome

displayed similar topologies overall (Fig 3) Among the four treesGPV-QH15 isolated from diseased ducklings was consistentlygrouped with GPVs rather than with MDPVs Furthermore GPV-QH15 clustered with GPVa2006 and GPV1995 two known goose-origin parvoviruses isolated from ducklings previously (2 6 817) to form the distinctive GPV IIa lineage PhylogeneticallyGPVa2006 and GPV1995 grouped most closely together withGPV-QH15 in all trees except for the viral NS1 tree The observeddifference in the NS1 tree was likely a consequence of geneticrecombination among parvoviruses in this particular gene Inter-estingly using RDP (Recombination Detection Program) (19) wedid not observe any significant recombination signals in this gene(data not shown) so the observed NS1-derived divergence war-rants consideration in future investigations The close associationof GPV-QH15 with GPVs at the genetic level is also consistentwith the result we obtained in the antigenic relationship studythrough a virus neutralization assay The antigenic analysisshowed that GPV-QH15 was more readily recognized and neu-tralized by antisera from a GPV than by those from an MDPVTaken together the phylogenetic analyses demonstrate that GPV-QH15 is more closely related to GPVs especially GPVa2006 andGPV1995 than to Muscovy duck parvoviruses

The structural modeling of the VP2 protein a major determi-nant of viral receptor binding and host specificity (20 21) furthersupported the close relationship observed between GPV-QH15on the one hand and GPVa2006 and GPV1995 on the otherStructure and protein homology analyses identified three variablesurface-exposed positions in the VP2 protein that are distinguish-able between MDPVs and GPVs VP2 positions 392 430 and 558were located in close proximity to the receptor-binding pocket(ldquocanyonrdquo) All MDPVs analyzed had I392 L430 N558 or L392K430 N558 while GPVs possessed L392 K430 D558 at these threepositions Significantly GPV-QH15 GPVa2006 and GPV1995differed from both MDPVs and other GPVs with an identicalpattern of amino acids I392 R430 N558 present at these posi-tions Taken together these findings support the supposition thatthe three viruses originated from a GPV lineage but now replicateand cause disease in ducklings The findings indicate a novel de-parture of these three parvoviruses from other GPVs and MDPVsin the biology of the VP2 protein viral evolution and probablyhost specificity It should be noted that VP2 N558 an invariantresidue observed among the three viruses and MDPVs likelyshifted GPV-QH15 GPVa2006 and GPV1995 toward the MDPVgroup These intriguing results support our antigenic relationshipstudy where we observed that the antigenic divergence betweenGPV-QH15 and MDPV was far less than that between GPV andMDPV in spite of the fact that GPV-QH15 was antigenically morerelated to GPV than to MDPV (Table 2) The amino acid differ-ences identified all located in the vicinity of the receptor-bindingpocket could possibly affect receptor binding and as a resultinfluence both host specificity and subsequent adaptation Thishypothesis warrants future experimental investigation

It has been well documented that both ducks and geese aresusceptible to GPV infection though only ducks can be infectedby MDPV (5 8 17 22ndash24) Little is known in the public literatureabout whether GPVa2006 and GPV1995 are capable of replicatingand causing disease in geese Since the initial description of GPV-QH15 in ducklings in October 2014 no instances of a tongueprotrusion-like syndrome have been documented in geese raisedon waterfowl farms where BADS outbreaks were found in duck-

TABLE 4 Detection of the viral genome in clinical tissuesa and fecalsamples

Group and sample typeNo of positive samplestotal screened ()

Ducklings from outbreak farmsWith DBADS

Tissue 3333 (100)Fecal swab 6382 (77)

With no clinical signsTissue 712 (58)Fecal swab 1538 (39)

Healthy ducklingsb 0100 (0)a Liver or spleen samples from ducklings on poultry farms with outbreaksb Samples were fecal swabs collected from healthy ducklings on poultry farms free ofparvovirus infection

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lings It is reasonable to speculate that through evolution goose-origin GPV-QH15 gained the ability to infect and replicate inducklings and as a result lost the ability to infect geese If this isthe case the amino acid variations identified in VP2 especiallythat at position 558 would likely be a primary determinant for thehost switch Further experiments are required to test this predic-tion

Despite the genetic and structural evidence that GPV-QH15GPVa2006 and GPV1995 are closely related to each other thethree viruses were different in terms of the disease severity causedin ducklings For example GPVa2006 could produce 100 mor-tality when inoculated into 1-day-old Muscovy ducklings but nei-ther tongue protrusion nor short-beak signs were reported forthese diseased animals (6) In contrast GPV-QH15 and GPV1995infections in ducklings resulted in tongue protrusion accompa-nied by smaller beaks with 3 to 6 mortality (2 8 17) Thetongue protrusion in ducklings infected with GPV-QH15 seemedto be more aggressive than that shown in GPV1995-infected ani-mals The VP1 gene of parvoviruses has been speculated to beinvolved in viral infectivity and pathogenesis Further analysis andbiological investigation of this gene and other components of theviral genome are needed to sort out this discrepancy in diseaseseverity and pathogenesis

It should be noted that during the preparation of this articleDiao and colleagues also reported on this emerging disease andisolated a causative parvovirus SDLC01 in China (15) Their clin-ical observations and genetic analysis were in good agreementwith many of our findings reported in this study Nevertheless bycarefully analyzing the full-length sequences of SDLC01 and ofGPV-QH15 reported here we came to realize that some differ-ences existed in the viral sequence largely focused on the 5= and 3=inverted terminal repeat (ITR) sequences For example we notedthree deletions in the ITR sequences of SDLC01 one deletionoccurring between positions 152 and 165 in the 5= end of the viralITR and two deletions occurring in the 3= end of the viral ITR onebetween positions 4823 and 4841 and one between positions 4890and 4903 These three deletions made the genome of our GPV-QH15 slightly longer than that of SDLC01 Further experimentsshould be pursued to confirm these sequence variations and toexplore their potential significance in the biology and antigenicityof these newly emerging duck parvoviruses in China

In summary the results of our experimental studies suggestthat GPV-QH15 represents a new variant of goose parvovirus thatcurrently circulates in ducklings and causes BADS This new find-ing highlights the need for future surveillance of GPV-QH15 inpoultry and for further investigation to better understand both theevolution and the biology of this emerging parvovirus

ACKNOWLEDGMENTS

We thank Ian Hauffe for reading and editing the manuscriptThis study was funded in part by the Shandong Modern Agricultural

Technology and Industry System (SDAIT-13-011-01) the major scien-tific and technological innovation project of the Shandong Academy ofAgricultural Sciences (2014CXZ08) the Shandong Provincial NaturalScience Foundation (BS2009YY019 Taishan Scholar Program to the In-stitute of Poultry Science) the Shandong Provincial Natural ScienceFoundation (ZR2015CM009) the Shandong Provincial Science andTechnology Development Plan Item (2013GNC11026) the Youth Scien-tific Research Foundation of the Shandong Academy of Agricultural Sci-ences (2014QNM15) the National Science and Technology Special Fund

of China (2012FY111000) and the 2015 provincial major animal epi-demic special funds of Shandong

FUNDING INFORMATIONThis work including the efforts of Kexiang Yu Xiuli Ma Lihong QiCunxia Liu Bing Huang and Feng Li was funded by Shandong Academyof Agricultural Sciences (2014CXZ08) This work including the efforts ofKexiang Yu Xiuli Ma Lihong Qi Cunxia Liu Bing Huang and Feng Liwas funded by Shandong Modern Agricultural Technology and IndustrySystem (SDAIT-13-011-01) This work including the efforts of KexiangYu Xiuli Ma Lihong Qi Cunxia Liu Bing Huang and Feng Li wasfunded by Natural Science Foundation of Shandong Province (NaturalScience Foundation of Shandong) (BS2009YY019)

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miological and diagnostic aspects with emphasis on type 2c Vet Micro-biol 1551ndash12 httpdxdoiorg101016jvetmic201109007

2 Zadori Z Stefancsik R Rauch T Kisary J 1995 Analysis of the completenucleotide sequences of goose and Muscovy duck parvoviruses indicatescommon ancestral origin with adeno-associated virus 2 Virology 212562ndash573 httpdxdoiorg101006viro19951514

3 Jansson DS Feinstein R Kardi V Mato T Palya V 2007 Epidemi-ologic investigation of an outbreak of goose parvovirus infectionin Sweden Avian Dis 51609 ndash 613 httpdxdoiorg1016370005-2086(2007)51[609EIOAOO]20CO2

4 Wozniakowski G Samorek-Salamonowicz E Kozdrun W 2012 Quan-titative analysis of waterfowl parvoviruses in geese and Muscovy ducks byreal-time polymerase chain reaction correlation between age clinicalsymptoms and DNA copy number of waterfowl parvoviruses BMC VetRes 829 httpdxdoiorg1011861746-6148-8-29

5 Poonia B Dunn PA Lu H Jarosinski KW Schat KA 2006 Isolationand molecular characterization of a new Muscovy duck parvovirus fromMuscovy ducks in the USA Avian Pathol 35435ndash 441 httpdxdoiorg10108003079450601009563

6 Shien JH Wang YS Chen CH Shieh HK Hu CC Chang PC 2008Identification of sequence changes in live attenuated goose parvovirusvaccine strains developed in Asia and Europe Avian Pathol 37499 ndash505httpdxdoiorg10108003079450802356979

7 Tsai HJ Tseng CH Chang PC Mei K Wang SC 2004 Genetic variationof viral protein 1 genes of field strains of waterfowl parvoviruses and theirattenuated derivatives Avian Dis 48512ndash521 httpdxdoiorg1016377172

8 Zadori Z Erdei J Nagy J Kisary J 1994 Characteristics of the genomeof goose parvovirus Avian Pathol 23359 ndash364 httpdxdoiorg10108003079459408419004

9 Guindon S Delsuc F Dufayard JF Gascuel O 2009 Estimating maxi-mum likelihood phylogenies with PhyML Methods Mol Biol 537113ndash137 httpdxdoiorg101007978-1-59745-251-9_6

10 Tamura K Stecher G Peterson D Filipski A Kumar S 2013 MEGA6Molecular Evolutionary Genetics Analysis version 60 Mol Biol Evol 302725ndash2729 httpdxdoiorg101093molbevmst197

11 Eswar N Webb B Marti-Renom MA Madhusudhan MS Eramian DShen MY Pieper U Sali A 2006 Comparative protein structure mod-eling using Modeller Curr Protoc Bioinformatics Chapter 5Unit 56httpdxdoiorg1010020471250953bi0506s15

12 Laskowski RA Moss DS Thornton JM 1993 Main-chain bond lengthsand bond angles in protein structures J Mol Biol 2311049 ndash1067 httpdxdoiorg101006jmbi19931351

13 Morris AL MacArthur MW Hutchinson EG Thornton JM 1992Stereochemical quality of protein structure coordinates Proteins 12345ndash364 httpdxdoiorg101002prot340120407

14 Woolfson DN Evans PA Hutchinson EG Thornton JM 1993 Topo-logical and stereochemical restrictions in beta-sandwich protein struc-tures Protein Eng 6461ndash 470 httpdxdoiorg101093protein65461

15 Chen H Dou Y Tang Y Zhang Z Zheng X Niu X Yang J Yu X DiaoY 2015 Isolation and genomic characterization of a duck-origin GPV-related parvovirus from Cherry Valley ducklings in China PLoS One 10e0140284 httpdxdoiorg101371journalpone0140284

16 Chen H Dou Y Tang Y Zheng X Niu X Yang J Yu X Diao Y 2016Experimental reproduction of beak atrophy and dwarfism syndrome by

Yu et al

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infection in Cherry Valley ducklings with a novel goose parvovirus-relatedparvovirus Vet Microbiol 18316 ndash20 httpdxdoiorg101016jvetmic201511034

17 Palya V Zolnai A Benyeda Z Kovacs E Kardi V Mato T 2009 Shortbeak and dwarfism syndrome of mule duck is caused by a distinct lineageof goose parvovirus Avian Pathol 38175ndash180 httpdxdoiorg10108003079450902737839

18 Tu M Liu F Chen S Wang M Cheng A 2015 Role of capsid proteinsin parvoviruses infection Virol J 12114 httpdxdoiorg101186s12985-015-0344-y

19 Martin DP Lemey P Lott M Moulton V Posada D Lefeuvre P 2010RDP3 a flexible and fast computer program for analyzing recombinationBioinformatics 262462ndash2463 httpdxdoiorg101093bioinformaticsbtq467

20 Rossmann MG 1989 The canyon hypothesis Hiding the host cell recep-tor attachment site on a viral surface from immune surveillance J BiolChem 26414587ndash14590

21 Tsao J Chapman MS Agbandje M Keller W Smith K Wu H Luo MSmith TJ Rossmann MG Compans RW Parrish CR 1991 The three-dimensional structure of canine parvovirus and its functional implicationsScience 2511456ndash1464 httpdxdoiorg101126science2006420

22 Wang S Cheng X Chen S Lin F Chen S Zhu X Wang J 2015Evidence for natural recombination in the capsid gene VP2 of Taiwanesegoose parvovirus Arch Virol 1602111ndash2115 httpdxdoiorg101007s00705-015-2491-2

23 Zhao H Xie Z Xie L Deng X Xie Z Luo S Huang L Huang J ZengT 2014 Molecular characterization of the full Muscovy duck parvovirusisolated in Guangxi China Genome Announc 2(6)e01249 ndash14 httpdxdoiorg101128genomeA01249-14

24 Zhu Y Zhou Z Huang Y Yu R Dong S Li Z Zhang Y 2014Identification of a recombinant Muscovy duck parvovirus (MDPV) inShanghai China Vet Microbiol 174560 ndash564 httpdxdoiorg101016jvetmic201410032

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