Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. ilaMescolini Giulia Europe? in Avian circulating of strains characteristics molecular on new is What 1 Falchieri Marco Cecchinato Mattia Cecchinato in Mescolini circulating Giulia Europe strains in metapneumovirus aMPV-B of Avian epidemiology of Molecular characteristics molecular on Europe? new strategies. correct is control towards the tool What address useful better a aMPV-B to be of can order characterization and analysis in molecular origin sequence applied the gene geographic routinely conclusion, G their In be through to should distant strains detected. and phylogenetically according field been diagnosis clades and cluster had vaccines to to between a belonged the tended differentiation showed the strains where strains continued and detected species and field has recently host strains, separately more the vaccine-derived clustered the of since Unlike that they irrespective ’80s as showed strains. since the classified tree older in strains vaccine phylogenetic were the appearance field commercial The from first analysed as live its strains. viruses classified from vaccine-derived with aMPV-B were evolve and identity of to 60% vaccines nucleotide frequently with remaining 40% identity high The appearance, The nucleotide showing first low Europe. detection. its and in of from related, licensed Europe area phylogenetically strains in geographic being evolved or the has period strains, with aMPV-B that vaccine-derived relation Likelihood that to Maximum clear Nucleotide demonstrated prior the a using X, collected strains. displaying reconstructed MEGA aMPV-Bs circulating strains, and in of extra-European aMPV-B the characteristics sequence implemented and among by molecular method European relationships analysed the Phylogenetic of were of sequences GenBank. (2014-2019), picture from other retrieved years updated with few an compared last obtained draw were B, the to subtype sequences in (aMPV) order metapneumovirus Countries in Avian European of analyses different sequences phylogenetic gene in G diagnostics partial sixteen routine and during hundred one study present the In Abstract 2020 19, May 7 6 5 4 3 2 1 Su˜n´e EAA EEUCRISTAL RESEAU CRISTAL – RESEAU Arag´oRESALAB – i Conseil Catalunya Av´ıcola VET de Sanitat MC de Centre - Agripolis CESAC di Campus Padova - of Padova university di Studi degli Universita available not Affiliation Bologna of University eateto eeiayMdclSine,Uiest fBlga zaodl’mla(O,Italy (BO), dell’Emilia Ozzano Bologna, of University Sciences, Medical Veterinary of Department 5 arzoTatone Fabrizio , 2 lui ai Tucciarone Maria Claudia , 6 ln Catelli Elena , 1 aeiaLupini Caterina , 1 aeiaLupini Caterina , 4 lui Tucciarone Claudia , 7 ac Falchieri Marco , 1 1, ,Goan Franzo Giovanni *, 2 neaBlanco Angela , 1 ivniFranzo Giovanni , 3 2 neaBlanco Angela , n ln Catelli Elena and , 1 3 icn Turblin Vincent , 2 ilaQuaglia Giulia , 2 ilaQuaglia Giulia , 5 icn Turblin Vincent , 1 4 a Biarn´es Mar , 1 atoLegnardi Matteo , 1 atoLegnardi Matteo , 3 6 arzoTatone Fabrizio , a Biarn´es Mar , 2 Mattia , 3 5 , , Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. Ctlie l,20;Cchnt ta. 03;Lsot ta. 04 rnoe l,21;Tcirn tal., et Tucciarone 2017; al., et Franzo scattered 2014; and al., poor et still Listorti are 2013a; Europe al., in et circulating Cecchinato strains in 2004; aMPV identified al., on were D, data et and 2006). molecular (Catelli study, C al., reports, initial named et this subtypes, these Toquin After further Despite 2000; two subtypes. Eterradossi, clas- analysis, both & the sequence of Toquin, proposed of co-circulation Arnauld, and use (Bayon-Auboyer, the isolates increasing France confirmed aMPV the and between to attenua- subtypes gene due live Europe, in G B and 1989b) in the problems and circulating e in A clinical strains 1989a differences into the analysing al. reported sification By and et (1994) available. Europe (Cook Easton commercially over 1990s and Rhinotracheitis became all Juhasz early and Ben- the developed spreading when (Giraud, were during was 1980s, France vaccines serious, virus and the ted increasingly the 1986) of became As Cook, half farms and 1986). second poultry Toquin, (McDougall the Kingdom & United to Guittet, the back nejean, in date appeared Europe outbreaks in (TRT) losses isolations secondary economic aMPV by causing exacerbated First 2018), often 2014). 2012), al., al., al., Catelli et et et 1999; (Giovanardi Cecchinato (Cecchinato problems infections 2017; Jestin, reproductive bacterial Catelli, & or & respiratory Eterradossi, to Bayon-Auboyer, Munir, due Ferreira, (Toquin, mainly Cecchinato, species 2001; minor al., other et in and chickens turkeys, metapneumovirus better Avian to order in applied Introduction routinely be should and cha- words diagnosis molecular Key sequence correct the gene towards G conclusion, strategies. through tool In control strains origin useful detected. the field geographic a been address and older vaccines their had be between the viruses to can differentiation from the according the analysis distant where and cluster phylogenetically aMPV-B species to of clades host racterization tended the to strains of belonged appearance irrespective field strains first and its strains, detected vaccine- from vaccine-derived recently and evolve vaccines Unlike to more as continued with strains. classified since has identity virus were ’80s nucleotide the 60% low the that remaining a showed viruses in showed tree The aMPV-B phylogenetic and Europe. The of separately in strains. nucleo- clustered 40% derived appearance, licensed high they The showing strains first since vaccine and detection. its strains related, commercial of field from phylogenetically live area being with Europe geographic identity strains, in vaccine-derived tide the evolved as method with classified has Likelihood relation were aMPV-B Maximum analysed clear Phy- the that GenBank. the a of from demonstrated using displaying retrieved X, picture reconstructed frequently or MEGA sequences strains, updated period aMPV-B in other an that the implemented to with draw prior among compared to collected were relationships order aMPV-Bs (2014- sequences logenetic extra-European in years Nucleotide and few analyses strains. last European phylogenetic the of circulating (aMPV) and in metapneumovirus of Countries e- sequence Avian characteristics European of by 2097559; molecular different sequences analysed in gene 051 diagnostics were G routine partial +39 2019), during sixteen Phone obtained and hundred B, Italy; one subtype study (BO) present the dell’Emilia In Ozzano Bo- – of 40064 -University Summary 50; Sciences Sopra, Medical Veterinary di mail: of Tolara Department Via Lupini, logna, Caterina author: *Corresponding 6 5 4 3 2 il eeiain ntdKingdom United Veterinarian, France Field Herbiers, Les CRISTAL, RESEAU France – Sarthe, Sabl´eRESALAB sur CRISTAL, RESEAU – Spain. Conseil Arag´o, Reus, VET Italy i MC (PD), Catalunya Av´ıcola Legnaro de Sanitat Padua, de of Centre University - Health, CESAC and Production Medicine, Animal of Department [email protected] va eanuoiu,sbyeB uoe oeua characterization molecular Europe, B, subtype metapneumovirus, Avian : aP)ifcin nErp aebe nisesneisfis pernebt in both appearance first its since issue an been have Europe in infections (aMPV) 2 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. lgmn as ihngopma ariegntcpdsac a siae sn EAXi re to order in X MEGA or using reliable. population. data Results estimated aMPV missing considered was containing of performing were p-distance sites heterogeneity by 70% genetic remove genetic calculated to pairwise the [?] was begins, evaluate mean analysis values support Within-group the branch bootstrap gaps. before by The selected alignment was supported 2018). option branches algo- deletion al., only neighbor-joining Complete et the replicates; (Kumar using bootstrap X reconstructed 1000 MEGA were in strains implemented aMPV-B rithm the among relationships from Phylogenetic retrieved - sequences vaccine S1) aMPV-B (Table aMPV/B extra-European sequences VCO3; 59 PL21) B - strain strain aMPV - - European vaccine vaccine available aMPV/B aMPV/B already and 82 1062 (i.e. - aligned strain vaccines - W, B vaccine subtype Clustal aMPV/B used 11/94; using Strain commonly then, most software, of: The sequences BioEdit - gene using G assembled with compared and and edited against were detection. sequences of number/year Nucleotide ID fowl)/sample analysis Guinea phylogenetic or and Ck Sequence aMPV/B/Country Chicken: Ty, nomenclature: (Turkey: following the species using Amsterdam, origin/Host named were Europe, of sequences (Macrogen obtained The (5’- service G9+B Netherlands). sequencing and The CAAAGAGCCAATAAGCCCA-3’) ExoSAP-IT commercial (5’- sequenced G5- a using and primers by instructions PCR TAGTCCTCAAGCAAGTCCTC-3’), purified RT-nested manufacturer’s using the were directions to both according obtained in USA) Massachusetts, amplicons Scientific, gene Fisher (Thermo G partial were The samples gene positive sequencing only G gene and RT-PCR the G real-time 2013b) multiplex Partial targeting al., a et RT-PCR by PCR. (Cecchinato preceded RT-nested nested gene was by step a differentiation SH amplified this the and subsequently to laboratory, targeting detection the subjected test simultaneous on screening Depending the and (qRT-PCR) B. allows swabs and which A of 1999) subtypes Naylor, of pool & each Britton, Mawditt, from (Cavanagh, extracted was (Catelli RNA suggested analysis previously PCR as and flock per extraction swabs RNA also rhino-pharyngeal was 10 of Sampling 2004). pools flocks. of al., fowl signs. consisted et guinea samples clinical rule, clear or a of respi- chicken As absence turkey, of in outbreaks in purposes during infections epidemiological collected Centre aMPV for usually the to performed were by and referable Spain countries disorders in European or ratory different Padua from and originated Bologna routine Samples during of Arag´o detected Universities (CESAC). B i the subtype Catalunya Av´ıcola aMPV by de targeted Sanitat Italy and de in 2019 to performed 2014 activity from diagnostic period time the covered survey The to designed collection the was Sample in study Europe in present Methods detected the and strains strains, Material B subtype circulating aMPV of sequencing, gene picture years. G last epidemiological partial 2011). the by in al., characterize, subtype update et encountered molecularly (Lupini frequently to most reported from order the originate sporadically generally them In been of been has most has A as B subtype 2019), subtype although al., decades, et Europe, last Andreopoulou the 2018; In Ganapathy, countries. & few Forrester, Ball, 2018; a 2017 3 GenBank TbeS2). (Table Express C rdc Cleanup Product PCR Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. o edsriscruaigi rzl rn saladTre.Vcie,vciedrvdadodrstrains older and subtrees. vaccine-derived two Vaccines, identified in compressed were Turkey. figure and clusters the Discussion Well-defined Israel in showed Iran, strains. were Brazil, confirmed and European in 2) together the (Figure circulating clustered world for strains the seen over field all trend for from clustering sequences Country-specific aMPV-B representative the with vac- reconstructed with tree clustering The VCO3. strains, vaccine-derived or displayed 11/94 exclusively PL21, S5) strain (Figure cine tree vaccine phylogenetic with British clustered strains The vaccine-derived p-distance: p- Spanish mean mean (within-group PL21. (within-group 2015. one or one to smaller larger 11/94 2014 a a strain from 2017; detected clusters: to main viruses 2014 two containing from within detected 0.003) fell strains strains containing field 0.006) in VCO3. S4) phylo- distance: detected (Figure or single all tree 11/94 Spanish a strains, PL21, vaccine-derived the within strains In The vaccine fell 0.001. chickens, with of clustered or p-distance flocks, turkeys mean turkey from within-group vaccinated turkeys. detected a vaccinated Vaccine-derived S3), showing from 0.004. group (Figure detected of genetic were strains p-distance 2001 1062 field mean from or aMPV-B within-group detected VCO3 a Romanian strains strains with field vaccine chickens, several and included aMPV-B and of flocks one turkeys with fowl composed in smaller clustering guinea was 2016 The strains and one in chicken, 0.011. turkey, larger and of in the 2009 2019 p-distance to S2): to mean (Figure 2010 within-group from clusters a detected main showed strains two were fowls. field in and guinea including grouped unvaccinated VCO3 sub-clusters detected were in or strains strains also 11/94 turkey but field PL21, chickens, contains Italian strain to or and vaccine 2017 turkeys 0.000 with homologous-vaccinated from of in respectively detected within- p-distance detected clustered strains a mean mostly fowl strains displayed within-group Vaccine-derived guinea cluster a and largest 2018. had turkey The in chicken, smallest included clusters. the two and while strains. in 0.004 2019, Greek divided of recent were p-distance field some S1) mean Italian with (Figure 46 group together and strains remaining French clustered field the Spanish, also aMPV Romanian, French strains (60%); among Italian strains observed Some same field was 1). the as clustering (Figure into geographic labelled strains of fell were degree they strains certain aMPV-B if strains. A vaccine-derived 116 and as of % identified out 99 were 70 (40%) to strains”. strains basis, equal sequence “field above nucleotide or as the maximum classified than the On were greater if strains was strains” remaining “vaccine-derived strain The as vaccine cluster. to reference phylogenetic forming referred a those were with and strains identity attenuation) aMPV by rule, established a 1990s, were As “recent the vaccines as to the to prior which detected referred (from strains herein clades. strains” field 2019, independent and “older to vaccines 2001 as with from to clustering detected referred those strains from aMPV distinguished origin, were of strains”, Country was five the sequences and of aMPV-B Regardless 1) extra-European and (Figure European 2). tree selected (Figure comprehensive including generated tree a phylogenetic sequences both a numbers S5). gene Furthermore, reconstructing to G accession S1 by partial under (Figures 116 out trees database 27 and specific carried GenBank n. Country- Kingdom) Italy, was the United from to analysis the 18 S7). submitted Phylogenetic from to n. 8 been France, S3 (Tables have from n. MT436220-MT436237 27 data and and Sequence (n. MT432826-MT432923 Spain B from subtype obtained. 36 to were belonging n. all Romania, positive, from aMPV tested samples 116 4 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. rnh tla,adSaihtessoe ahrhtrgnosfil tanpplto,a h tan fell strains the as population, strain 2020). field different al., heterogeneous from et rather (Franzo came a compartmentalized samples showed be This trees processed could clade. Spanish the circulation one and all viral just Italian, that which French, of account in part company into were same keeping strains the by identified within explained country- all European sites be where reconstructing single Romania, could within detail of co-exist finding in to exception last analysed seemed only was populations the strain country with field countries, each Heterogeneous within trees. aMPV some phylogenetic 2019). between related of yet al., route epidemiology et strains, transmission molecular Andreopoulou potential Spanish 2017; The a and al., indicating Romanian few et together, (Tucciarone with closely Italian, countries origin, clustered French, two geographic isolates these for their field observed Greek to were and according Italian clusters cluster to phylo- Distinct tended clades strains to exceptions. field evolve belonged to strains, strains vaccine-derived tendency field Unlike aMPV detected 2010). previously-reported recently al., the more et confirming (Cecchinato fact, strains, evolve time field In to over older continued ’80s. the has the from virus distant in the genetically that appearance they showed first strains. since sequences vaccine-derived its strains European and from with field vaccines reconstructed as with tree classified identity phylogenetic were nucleotide The study low present a aMPV the showed by in and caused analysed separately crops outbreak viruses clustered rhinotracheitis surrounding aMPV-B turkey the 60% a known of remaining following well applied fertilization The (2011) is was for site al. turkeys. farm administration 11/94 unvaccinated et the turkey in Lupini strain beyond the by A Vaccinal spread proven from subtype to been litter one. aMPVs has the vaccine-derived fowl fomites, it of of guinea and personnel, ability use of the by The the presence from contaminant reported. and the meters was a flock reported 500 veterinarian turkey as approximately field the introduced The at in could been It farm premises. had neighbouring turkey 11/94. could strain from a vaccine virus airborne the or vaccine with movement identity the nucleotide vehicles 100% that notewor- the shared speculated is sequence to fowls gene be linked guinea G unvaccinated partial reliably in its aMPV/B/France//1060/18 be since strain thy, could strains the vaccine-derived of signs, of (Catelli detection respiratory detection The demonstrated the with previously homologous- Therefore, concurrently been virulence. vaccine, 2014). in has to al., applied unvac- detected reversion B et the vaccine in Cecchinato or was to occasionally 2011, A study al., related only subtype et present and closely aMPV Brown vaccination, of the 2006; after virulence al., in weeks to et analysed four Reversion to strains birds. two vaccine-derived cinated approximately the from of birds, of strain vaccinated part vaccine countries. the European large on tested A depending licensed all clusters strains in separate vaccine detected formed commercial were strains live being and vaccine-derived et strains, with origin expected, vaccine-derived Arafa identity as As 2014; classified nucleotide were al., Europe. high study et in present showing Listorti the and in 2013a; related detected al., strains 2019). phylogenetically et aMPV-B al., the Cecchinato et of 2011; 40% Andreopoulou al., The 2019; (Banet-Noach et al., flocks Chacon unvaccinated et 2011; the in Bayraktar al., and or 2015; chickens, vaccinated et al., and in Lupini turkeys both 2009; in unusual al., disease not et prevent is perform. to strains proved to administered vaccine-derived sequencing cheap of widely recovery are and or its vaccines vaccine easy and aMPV whether relatively attenuated of study, conveniently differentiation Live indication the was the an making give strains, in present, can employed aMPV-B variability are its strains between protocol Furthermore, field RT-PCR positions purposes. variable epidemiological diagnostic for at routine useful mutations the harbours by which amplified routine gene, phylogenetic during and G obtained sequence them. by sequences, The characterize analysed gene were molecularly years, G to few partial order last aMPV-B in the in sixteen analyses Countries and European different hundred in one diagnostics study, present the In 5 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. tia approval interest. Ethical of conflict no declare authors aMPV The modified study foreign Statement rationally the expressing Interest of for 2013) development of tool al., the Conflict exceptional et for (Falchieri an vaccines used being recombinant be than subtypes or can other C 2010) 2011), 2016) and Brown, genes. still al., B & al., vaccines et Lupini, A, et live (Naylor, (Brown for Laconi vaccines years effort, properties 2010; recent al., this virus in et the despite developed Yu of but, been 2004; have 2017) al., aMPV Yu, et for (Naylor & systems Zsak, genetics reverse Roth, challenge. Promising Hu, Njenga, homologous Chary, after 2013; 2004; protection Kapczynski, al., greatest 2003; et the Sellers, Yu and provide Kapczynski vaccines 2005; 2001; next-generation Sharma, al., safer et and & to Tarpey stable 1994; more order al., efficacious, in et develop (Qingzhong to measures, attempted biosecurity have groups and research associated administration Several spreading. the the improving and and or considering at diagnosis circulation virulence Therefore, directed prolonged to correct vaccine 2015). their be reversion a current vaccine reduce al., should towards respect, attenuated et efforts live this (Franzo tool further In to subpopulations useful risks, connected strategies. virulent a issues control potentially the the be of address reducing selection can vaccines better on between to analysis focused order differentiation is sequence in the research gene applied and routinely G B be through subtype should aMPV phylogenetically-distant strains of of field characterization clusters aMPV and molecular two homogeneous the of quite conclusion, exception subtree a single In only big of a the a and circulation after with in strains. wide location place countries, located Spanish the taken country European were and French have supporting in strains for might and clade field together that similarity B European grouped virus subtype genetic detected the strains the recently certain confirmed of of field a further tendency majority indicating rule, strains evolution The a extra-European local event. As and a introduction European suggesting strains. on period, field reconstructed evidences time by of tree no caused Therefore, the clustering variants. fowls remark, geographic aMPV flocks. guinea final of turkey in adaptation both a surrounding disease host-specific As the 2019). that a respiratory in al., showed support of et circulating to study (Brown outbreak ones available isolate challenge currently an the virus are experimental to reported same identical recent (2018) the host strain with A the aMPV-B al. infection of aMPV-B et detected. irrespective to Cecchinato together susceptible been Moreover are clustered had turkeys strains and viruses aMPV-B chickens the disease that improve where revealed to data species necessary phylogenetic variability be obtained might gene The strains G field commercially the antigen aMPV by conferred key vaccine-induced to strategies. circulating protection avoid a control of currently to Due re-assessment is order 2010), against glycoprotein in al., vaccines evolve G et flocks. can available Cecchinato surface and 2010; vaccinated encoded al., 2007) et in al., the clades, (Catelli et immunity that phylogenetic signs (Naylor knowing distant protection respiratory and immune and vaccine-induced strains, different of for aMPV-B to presence recent belonging in the strains, observed aMPV-B in in field detected detected sub-clusters. recent were strains or finding, sequences clusters as common separate of strains, a form field As number to Spanish tend larger and years companies. Italian the few poultry for last of commercial visible the different was because from clustering presumably coming time-related and nucleotide Furthermore, strains, period of field time the heterogeneity from broader highest Italian deduced a the for over further indicating available was value, observed heterogeneity highest was The The sub-clusters. sequence, values. several p-distance in divided mean cluster, within-group one than more into 6 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. er,F,Lsot,V,Msoii . oadn,E,...Ctli .(08.Mlclrcaatrzto of characterization Molecular Listorti, (2018). M., E. Martini, Catelli, C., Lupini, M., molecular . Drigo, . M, . meleagridis). Sil- Cecchinato, 2: vaccine: (numida E., https://doi.org/10.29261/pakvetj/2018.088 Volume fowls C., Morandini, metapneumovirus 423. Guinea Lupini, G., avian from importance. Mescolini, M., metapneumovirus E. B veterinary V., avian Cecchinato, Catelli, progenitors? Listorti, subtype of https://doi.org/10.1079/9781780644172.0127 & F., vaccine Mononegaviruses a veira, M., In of avian control. of Munir, availability and and virulence metapneumovirus. L., epidemiology require virus to Avian H. to bronchitis Reversion Ferreira, (2017). regulators infectious M., Prosperi, for of (2014). Cecchinato, E., https://doi.org/10.1016/j.vaccine.2014.06.030 time J. studies Ricchizzi, of it C. field C., Demonstration Lupini, Is Naylor, Longitudinal reactions. E., & (2006). chain vaccine. Catelli, J. S., (1999). polymerase metapneumovirus M., C. type-specific J. avian Cecchinato, using Naylor, live https://doi.org/10.1080/03079459994399 C. broilers & a Naylor, in C., of & pneumovirus R. persistence P., (2004). serologi- Jones, field C. and ton, E., extended Naylor, molecular C. and John virological, 24 Savage, attenuation a . of M., . Italy: . loss Cecchinato, in C., farms and E., Franciosi, broiler P., reared Catelli, and in Matteo, survey. turkey infection De field in pneumovirus cal M., infection avian Delogu, Pneumovirus of M., Avian evidence Cassandro, Serological M., Cecchinato, (2001). increased V. metapneumovirus pheasants. avian temperature. Guberti, free-living in elevated & mutation an C., (L) at Terregino, polymerase viability single virus A 92 maintained ogy, (2011). metapneumoviruses. partially J. avian and C. of virulence Naylor, specificity & Host E., Metapneumovirus chizzi, Avian (2019). non-A/non-B of N. Characterization two Eterradossi, 318. Molecular of First . . genes . (2019). protein H. G Flocks. Yilmaz, resnote.1 Broiler G., and Turkish Franzo, . in N., L . metapneu- Turan, . (aMPV) F, A., avian M., Yilmaz, the C. of S., Tucciarone, of Umar, subgroup. survey flocks. E., APV sequences novel vaccinated Bayraktar, Longitudinal a https://doi.org/10.1099/0022-1317-81-11-2723 Nucleotide into reveal (APV) strains pneumoviruses (2009). field avian (2000). E. of N. problems. infiltration res- Bacharach, Eterradossi, production unrelated & Israel: or and in S., health 184–189. related poultry Perk, with vaccine in flocks N., of moviruses poultry population Laham-Karam, UK diverse Vaccine- L., in genetically of 225 viruses Microbiology, Egypt. Characterization of nary and in Molecular Co-circulation mycoplasmas Turkeys M., piratory metapneu- (2015). from (2018). Cecchinato, avian H. Isolated K. Madbouly, and C., thy, 4 Meta-Pneumoviruses & virus K. Virology, Avian S., bronchitis of of Koutoulis, Tamam, infectious Mutants Z., A., of Derived Hussein, Prentza, epidemiology W., Molecular M., Mady, Greece. C. (2019). in Tucciarone, movirus I. G., Chaligianni, Franzo, & M., Andreopoulou, reasonable upon author corresponding the References from available was are article study this this of findings of request. the data support that research data original veterinarians. The field the by as purpose Statement page, required diagnostics Availability guidelines routine was author Data for approval journal’s the collected ethical on swabs No noted oro-pharyngeal as from journal, to. obtained the adhered of policies been ethical have the that confirm authors The 4-3,67–42 tp:/o.r/011/.acn.060.7 aaah . adt,K,Brit- K., Mawditt, D., Cavanagh, https://doi.org/10.1016/j.vaccine.2006.06.076 6476–6482. (42-43), https://doi.org/10.1080/03079457.2019.1584390 P ) 4–5.hts/diog1.09vr0064- ael,E,D ac,M . eou M., Delogu, A., M. Marco, De E., Catelli, https://doi.org/10.1099/vir.0.026740-0 346–354. 2), (Pt https://doi.org/10.1637/8466-090408-Reg.1 rw,PA,Ale . oriln . zra,N,Lmir,E,Tqi,D,Mnat J.M., Mangart, D., Toquin, E., Lemaitre, N., Szerman, C., Courtillon, C., Allee, P.A., Brown, 1,11.hts/diog1.84avp21..1Bl,C,Fretr . Ganapa- & A., Forrester, C., Ball, https://doi.org/10.3844/ajvsp.2015.1.11 1–11. (1), tla ora fAia cec,3 Science, Animal of Journal Italian olr cec,98 Science, Poultry 3–3.hts/diog1.06jvti.080.0 ae-oc,C,Simanov, C., Banet-Noach, https://doi.org/10.1016/j.vetmic.2018.09.009 132–138. , eeiayRcr,149 Record, Veterinary va iess 62 Diseases, Avian 1) 3458.hts/diog1.32p/e30Aaa A.-S., Arafa, https://doi.org/10.3382/ps/pez360 5374–5384. (11), 2,5–8 tp:/o.r/013/r1925 ael,E., Catelli, https://doi.org/10.1136/vr.149.2.56 56–58. (2), ao-uoe,M . ral,C,Tqi,D,& D., Toquin, C., Arnauld, H., M. Bayon-Auboyer, rw,P . uii . ael,E,Cub,J,Ric- J., Clubbe, E., Catelli, C., Lupini, A., P. Brown, 7 3,2722 https://doi.org/10.4081/ijas.2004.287 287–292. (3), 4,425–430. (4), ora fGnrlVrlg,81 Virology, General of Journal aitnVtrnr ora,38 Journal, Veterinary Pakistan https://doi.org/10.1637/11915-061818- va ahlg,28 Pathology, Avian va ahlg,48 Pathology, Avian acn,32 Vaccine, ora fGnrlVirol- General of Journal va iess 53 Diseases, Avian mrcnJournal American 3) 4660–4664. (36), 1) 2723–2733. (11), 6,593–605. (6), 4,311– (4), 4,419– (4), Vaccine, Veteri- (2), Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. . lbe . acir,M,Lpn,C,Cchnt,M,Ctli . alr .J 21) comparison A (2016). J. plat- C. Naylor, computing across . . . analysis E., Catelli, genetics M., evolutionary Cecchinato, C., Molecular Lupini, Metapneumovirus. M., X: Falchieri, Avian J., MEGA of Clubbe, Gene A., N (2018). or K. F forms. Tamura, the Either & Expressing C., Vaccine 47 Turkeys. DNA Diseases, in (G) a Protection Avian attachment with for Turkeys the C of Subtype in nization Metapneumovirus variation subgroups. Avian 48 distinct sequence Against eases, two Vaccine Extensive Virosome for a Evidence (1994). of confers J. pneumovirus: AMPV-C A. avian of 75 proteins of Easton, G gene & and protein F virus. K., the ciliostatic turkeys. Juhasz, expressing a virus in 017-04267-7 on disease challenges Newcastle investigations against Engineered preliminary protection (2017). France: Q. in Yu, & rhinotracheitis en- Turkey hemorrhagic 119 Turkey Record, (1986). Veterinary and D. metapneumovirus mortality. Toquin, Avian associated & colibacillosis of from studies 38 suffering G., field tions, turkeys Ortali, Longitudinal en- in G., and virus farms. Rossi, (2014). anthropic teritis P., poultry E. between Pesente, Italian Catelli, balance C., the & among Lupini, of spreading D., evaluation Giovanardi, IBV https://doi.org/10.1038/s41598-020-64477-4 and M. affecting analysis Cecchinato, factors Phylodynamic Dur- & Flock vironmental C., Broiler (2020). Romanian K. M. a Koutoulis, Cecchinato, in Disease. G., Strain Respiratory Field Ramon, of B only 121216-ResNote.1 V., the Subtype Outbreak Metapneumovirus be Bejan, an to Avian M., ing unlikely of Enache, are Report vaccines First M., aMPV C. (2017). in Subpopulations Tucciarone, virulence. Ducatez, (2015). G., M. to Franzo, G., Cecchinato, reversion Croville, of . M., . . cause Drigo, E., protection. J., Catelli, metapneumoviruses induce F., C. Avian and M. Naylor, (2013). stable Live J. G., Experimen- are C. A Franzo, an genes Naylor, (1989b). https://doi.org/10.1016/j.vaccine.2013.03.055 & as virus M. M., Bronchitis Strain P. Kontolaimou, Finney, Attenuated Infectious E., & the expressing Catelli, C., M., of H. Cecchinato, Use Holmes, C., B., the Lupini, M. 2. Huggins, Vaccine. M., Vaccine. M. tal Virus Ellis, Rhinotracheitis A., Stability Turkey C. 1. Dolby, Attenuated Vaccine. A., K. Virus J. Rhinotracheitis Turkey Strain. Cook, Attenuated Attenuated Live the A of (1989a). chal- P.M. respiratory Finney, a H.C., against Holmes, proteins J. viral recombinant A. by Ferreira, metapneumovirus. Protection 432. Piantino avian (2005). virulent M. with J. . lenge . Sharma, Chicken . & Nonvaccinated P., K., and M. D. Vaccinated Njenga, Patnayak, Brazilian Farms. N., in Turkey Reveals Circulating Eterradossi, Survey and Subtypes D., Strat- Metapneumovirus Field Toquin, Vaccination Avian Italian P., the in (2011). M. (2012). Weaknesses Vejarano, E. and Layers M., Catelli, for in & Mizuma, assay B C., Subtype RT-PCR R. Applied. Metapneumovirus real-time Jones, Avian egy a of A., Diffusion Meini, of High M., Development and a Falchieri, A E., (2013b). subtypes Ricchizzi, metapneumovirus E. C., avian Catelli, of differentiation & Lis- and B. M., quantitation S., Drigo, identification, O. simultaneous A., Pogoreltseva, the Mondin, Munoz probable V., C., Italy. in involvement introduction. Northern Lupini, torti, protein vaccine in M., attachment live metapneumovirus (AMPV) Cecchinato, mass avian metapneumovirus https://doi.org/10.1016/j.vetmic.2010.04.014 with Avian of concurrent survey (2010). evolution Field J. virus C. (2013a). Naylor, & E. M., Catelli, 19 Review, . . Animal . G., Franzo, V., va ahlg,42 Pathology, Avian 1) 8328.hts/diog1.0902-377-127 acysi .R 20) Development (2004). R. D. Kapczynski, https://doi.org/10.1099/0022-1317-75-11-2873 2873–2880. (11), https://doi.org/10.1016/j.vetimm.2005.06.012 oeua ilg n vlto,35 Evolution, and Biology Molecular 2,1917 tp:/o.r/010/1290499- iad . enja,G,Gitt M., Guittet, G., Bennejean, P., Giraud, https://doi.org/10.1007/s11259-014-9596-z 129–137. (2), 2,3233 tp:/o.r/013/15Kpznk,D . elr,H .(03.Immu- (2003). S. H. Sellers, & R., D. Kapczynski, https://doi.org/10.1637/7115 332–343. (2), va ahlg,18 Pathology, Avian va iess 56 Diseases, Avian rno . ucaoe .. oeo . enri . as,P,Ts,G,..., . . . G., Tosi, P., Massi, M., Legnardi, A., Moreno, C.M., Tucciarone, G., Franzo, 6,2720 eciao . ael,E,Lpn,C,Rchzi . lbe . Battilani, J., Clubbe, E., Ricchizzi, C., Lupini, E., Catelli, M., Cecchinato, 267–270. (6), 4,17–33 tp:/o.r/013/03Kmr . tce,G,L,M,Knyaz, M., Li, G., Stecher, S., Kumar, https://doi.org/10.1637/7033 1376–1383. (4), va iess 55 Diseases, Avian 3,2329 tp:/o.r/018/374721.810Cchnt,M,Lupini, M., Cecchinato, https://doi.org/10.1080/03079457.2013.788130 283–289. (3), 2) 0–0.hts/diog1.16v.1.466aH,H,Rt,J . sk L., Zsak, P., J. Roth, H., Hu, https://doi.org/10.1136/vr.119.24.606-a 606–607. (24), va ahlg,18 Pathology, Avian acn,33 Vaccine, 4,7074 tp:/o.r/013/00-432rg1Cao,J L., J. Chacon, https://doi.org/10.1637/10202-041312-reg.1 720–724. (4), 3,5354 tp:/o.r/018/37480482 acir,M., Falchieri, https://doi.org/10.1080/03079458908418624 523–534. (3), 1,8–9 tp:/o.r/013/51011-e. hr,P., Chary, https://doi.org/10.1637/9501-081310-reg.1 82–89. (1), 6,14–59 tp:/o.r/019/obvmy9 Laconi, https://doi.org/10.1093/molbev/msy096 1547–1549. (6), cetfi eot,7 Reports, Scientific va iess 61 Diseases, Avian 2) 4824.https://doi.org/10.1016/j.vaccine.2015.03.092 2438–2441. (21), eeiayImnlg n muoahlg,108 Immunopathology, and Immunology Veterinary 3,5152 https://doi.org/10.1080/03079458908418623 511–522. (3), ok ... li,MM,Dly .. ugn,M.B., Huggins, C.A., Dolby, M.M., Ellis, J.K.A., Cook, 8 2,250–254. (2), 1,42.https://doi.org/10.1038/s41598- 4025. (1), eeiayMcoilg,146 Microbiology, Veterinary eeiayRsac Communica- Research Veterinary https://doi.org/10.1637/11557- ora fGnrlVirology, General of Journal cetfi eot,10 Reports, Scientific acn,31 Vaccine, 2) 2565–2571. (22), 12,24–34. (1–2), va Dis- Avian 34,427– (3-4), 7289. , Large Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. rgn h onr foii a de nbakt ett h tannm hni a o lal specified clearly not reported. was are it of when 70 country name [?] by strain values the coloured bootstrap to are next Only brackets clusters name. in strains very added was the and aMPV-B origin vaccines in Field of containing country subtrees The two compressed. The origin. been GenBank. have from strains retrieved or vaccine-derived reported. obtained are previously 70 strains, European [?] aMPV-B values 82 bootstrap brackets and 2. square Only vaccines in Figure origin. included and B of are vaccines clusters country containing subtype strains by subtrees aMPV-B used two coloured Field The and commonly compressed. GenBank. most been from have retrieved the strains or vaccine-derived including obtained previously study, strains, present aMPV-B the in detected 1. Figure Virulent with Challenge against Expressing legends B (NDV) Figure or Viruses A Disease Subtype Newcastle (aMPV) Recombinant aMPV. Metapneumovirus by and Avian Protection NDV of (2013). avian (G) L. recombinant Glycoprotein Zsak, of the & gene. assessment G W., biological the Zhao, Italian and of N., in Generation length different circulation (2010). containing Metapneumovirus viruses 147 L. Avian (aMPV-C) Zsak, C & (2018). subgroup D., M. metapneumovirus Cecchinato, Kapczynski, M., . Song, . . C. Molecular G., Alejo, farms. and C., Mescolini, broiler Identification Greece. Lupini, V., First in G., Franzo, Listorti, Chickens (2017). M., T., from C. M. B Tucciarone, Cecchinato, https://doi.org/10.1637/11631-032017-CaseR Subtype & 409–413. metapneumovirus I., Avian Chaligiannis, lineages. of Z., genetic Characterization Prentza, different to G., belong Franzo, metapneumovirus M., A. avian 32 of K. duck. isolates Genes, Muscovy J. C Virus a subgroup Cook, from protein. American pneumovirus & and a F of European J., isolate) Isolation S. (Colorado (1999). V. 23 Orbell, pneumovirus Record, Jestin, avian R., & N. the Shilleto, Eterradossi, of M.H., 471–474. J., D. immunogenicity Cavanagh, P. 30(5), and Pathology, & Davis, expression A., Avian B., M. Cloning, M. Skinner, P., Huggins, (2001). recombi- Green, I., virus A., (F). fowlpox Tarpey, glycoprotein a K. 410X(94)90319-0 by fusion J. TRTV induced (TRTV) Cook, the pneumovirus expressing K., rhinotracheitis nant D. turkey than against T. protection Protection Brown, induced on (1994). T., genes. influence Barrett, more G have Y., or protein Qingzhong, SH fusion AMPV the the candidates. of in live-vaccine acids deletion of amino Charged protection gene of SH (2010). metapneumovirus level A. Avian the (2007). influence J. A. 88 mutations Virology, Easton, for protein & system E., G reverse-genetics C. and Savage, a N., end Edworthy, of R., Development Ling, essential J., not (2004). C. are Naylor, J. genes (G) A. attachment and Easton, viability. (SH) virus & hydrophobic for small E., the C. that Savage, demonstrates pneumovirus C., investigations. Avian R. preliminary Jones, rhinotracheitis: Turkey R., Ling, (1986). K. J. 118 Cook, Record, vaccine-derived & a (2014). of S., & spread J. environmental E. J., McDougall, the by Catelli, C. diges- caused Naylor, outbreak metapneumovirus. endonuclease rhinotracheitis E., avian turkey . turkeys. . restriction A Ricchizzi, . older RT-PCR (2011). M., D., in E. Cecchinato, using Catelli, Giovanardi, viruses C., vaccine-derived metapneumoviruses G., of Lupini, avian Rossi, https://doi.org/10.1080/03079457.2013.866212 circulation P., B field Pesente, indicates subtype res- M., systems tion of reverse-genetics Cecchinato, to detection led C., Rapid proteins Lupini, and transcripts V., gene Listorti, genomes, full subtypes. B both and cuing A subtypes AMPV of 2,1218 tp:/o.r/011/.iurs20.001Y,Q,Rt,J . u . see,C. Estevez, H., Hu, P., J. Roth, Q., Yu, https://doi.org/10.1016/j.virusres.2009.10.021 182–188. (2), 8.Tqi,D,Gine . etn . wnesen . le,C,&Eerdsi .(2006). N. Eterradossi, & C., Allee, F., Zwingelstein, V., Jestin, O., Guionie, D., Toquin, 680. , hlgntcte ae nGgn uloiesqecso eetdErpa n extra-European and European selected of sequences nucleotide gene G on based tree Phylogenetic 8,2627 tp:/o.r/013/r18826Nyo,C . rw,P . doty N., Edworthy, A., P. Brown, J., C. Naylor, https://doi.org/10.1136/vr.118.8.206 206–207. (8), P ) 7717.hts/diog1.09vr08750Nyo,C . uii . rw,P. Brown, & C., Lupini, J., C. Naylor, https://doi.org/10.1099/vir.0.82755-0 1767–1775. 6), (Pt hlgntcte ae nGgn uloiesqecso 1 uoenaP- strains aMPV-B European 116 of sequences nucleotide gene G on based tree Phylogenetic olr cec,97 Science, Poultry 1,9–0.hts/diog1.07s16-0-803Tcirn,CM,Andreopoulou, C.M., Tucciarone, https://doi.org/10.1007/s11262-005-5850-3 97–103. (1), ol ora fVcie,03 Vaccines, of Journal World ora fGnrlVrlg,85 Virology, General of Journal ora fGnrlVrlg,97 Virology, General of Journal va ahlg,40 Pathology, Avian acn,28 Vaccine, https://doi.org/10.1080/03079450120078653 2,5359 tp:/o.r/038/spx5 u . see,C., Estevez, Q., Yu, https://doi.org/10.3382/ps/pex350 503–509. (2), 4) 8060.https://doi.org/10.1016/j.vaccine.2010.07.015 6800–6807. (41), 5,5550 https://doi.org/10.1080/03079457.2011.607428 525–530. (5), 0) 130–139. (04), P 1,31–27 https://doi.org/10.1099/vir.0.80229-0 3219–3227. 11), (Pt acn,12 Vaccine, 9 6,12–32 https://doi.org/10.1099/jgv.0.000450 1324–1332. (6), https://doi.org/10.4236/wjv.2013.34018 6,5953 https://doi.org/10.1016/0264- 569–573. (6), va ahlg,43 Pathology, Avian oun . Bayon-Auboyer, D., Toquin, va iess 61 Diseases, Avian ora fGeneral of Journal iu Research, Virus h Veterinary The h Veterinary The 1,51–56. (1), (3), Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. n 0BiihaP Bsris rvosyotie rrtivdfo eBn.Ol otta aus[?] values bootstrap Only triangle) green GenBank. a from with retrieved (marked or vaccines obtained B previously reported. subtype strains, are used -B 70 commonly aMPV most British reported. the 10 are including and 70 Field study, [?] present triangle). values the green bootstrap a in Only with (marked brackets. S5. vaccines square B Figure in subtype included used green are commonly a clusters most strains are the with and aMPV-B 70 (marked study [?] present vaccines values the B in bootstrap Only subtype brackets. S4. used Figure square commonly in included most are the clusters and strains reported. aMPV-B study strains Field present aMPV-B triangle). reported. Field the are 70 in GenBank. [?] detected from values triangle) retrieved bootstrap green S3. or Only a Figure obtained with brackets. (marked square previously vaccines in strains, B included subtype -B are used aMPV clusters commonly Italian most 42 the square including and in study, present included the are in clusters triangle) reported. strains green S2. are aMPV-B a 70 Figure with Field (marked [?] vaccines values obtained. B bootstrap previously subtype Only strains used brackets. aMPV-B commonly French most 2 the including and study, present the in S1. Figure legends figure Supplementary hlgntcte ae nGgn uloiesqecso 6SaihaP- tan detected strains aMPV-B Spanish 36 of sequences nucleotide gene G on based tree Phylogenetic hlgntcte ae nGgn uloiesqecso 7Fec MVBsrisdetected strains aMPV-B French 27 of sequences nucleotide gene G on based tree Phylogenetic hlgntcte ae nGgn uloiesqecso 8IainaP- tan detected strains aMPV-B Italian 18 of sequences nucleotide gene G on based tree Phylogenetic hlgntcte ae nGgn uloiesqecso rts MVBsrisdetected strains aMPV-B British 8 of sequences nucleotide gene G on based tree Phylogenetic hlgntcte ae nGgn uloiesqecso 7Rmna MVBstrains aMPV-B Romanian 27 of sequences nucleotide gene G on based tree Phylogenetic 10 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. 11 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. 12 Posted on Authorea 19 May 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.158990363.30460690 — This a preprint and has not been peer reviewed. Data may be preliminary. 13