1 Tracing the Flight: Investigating the Introduction of Avian Metapneumovirus (aMPV) A and

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2 B

3 Giovanni Franzo1*, Matteo Legnardi1, Giulia Faustini1, Riccardo Baston1, Francesca Poletto1,

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4 Mattia Cecchinato1, Claudia Maria Tucciarone1.

6 1Department of Animal Medicine, Production and Health, University of Padova, Legnaro, Italy

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7 *Corresponding author: giovanni.franzo@unipd.it

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8 Abstract

9 Avian metapneumovirus (aMPV) has been identified as an important cause of respiratory and
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10 reproductive disease, leading to significant productive losses worldwide. Different subtypes have

11 been found to circulate in different regions, with aMPV-A and B posing a significant burden
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12 especially in the Old World, and aMPV-C in North America, albeit with limited exceptions of

13 marginal economic relevance. Recently, both aMPV-A and aMPV-B have been reported in the
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14 U.S., however, the route of introduction has not been investigated. In the present study, the potential

15 importation pathways have been studied through phylogenetic and phylodynamic analyses based on
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16 a broad collection of partial attachment (G) protein sequences collected worldwide. aMPV-B

17 circulating in the U.S. seems to be the descendant of Eastern Asian strains, which, in turn, are
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18 related to European ones. A likely introduction pathway mediated by wild bird migration through

19 the Beringian crucible, where the East Asian and Pacific American flight paths intersect, appears

20 likely and was previously reported for avian influenza. aMPV-A, on the other hand, showed a
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21 Mexican origin, involving strains related to Asian ones. Given the low likelihood of trade or illegal

22 importation, the role of wild birds appears probable also in this case, since the region is covered by
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23 different flight paths directed in a North-South direction through America. Since the information on

24 wild bird role in aMPV epidemiology is still scarce and scattered, considering the significant

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059
25 practical implications for the poultry industry demonstrated by recent U.S. outbreaks, further

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26 surveys on wild birds are encouraged.

27 Keywords: aMPV; North America; Wild Birds; Phylogeography; Molecular Epidemiology

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28 Main text

29 Avian metapneumovirus (aMPV) is a highly contagious pathogen that affects turkeys and chickens,

30 causing turkey rhinotracheitis (TRT) and swollen head syndrome (SHS), respectively. aMPV has

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31 also the capacity to infect other avian species (Graziosi et al., 2022a; Tucciarone et al., 2022; Umar

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32 et al., 2016). The virus is primarily associated with upper respiratory infections, predisposing birds

33 to secondary bacterial pathogens, which can result in severe respiratory clinical signs, high

34
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morbidity, and mortality rates. Furthermore, aMPV can infect the reproductive system, leading to a

35 significant decrease in egg production (Salles et al., 2023).

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36 aMPV is identified as a single-stranded, non-segmented, enveloped, and negative-sense RNA virus,

37 with a size range of approximately 13.3–14 kb, belonging to the family Pneumoviridae and genus

38 Metapneumovirus. Its genome comprises eight genes arranged in the 5’-3’ direction as
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39 follows:Nucleoprotein (N), Phosphoprotein (P), Matrix (M), Fusion (F), Matrix 2 (M2), Small

40 Hydrophobic (SH), Attachment (G), and Large Polymerase (L), with leader and trailer sequences at
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41 both ends (3′-le–N–P–M–F–M2–SH–G–L–tr-5′) (Easton et al., 2004).

42 The attachment (G) and fusion (F) proteins are major structural components located on the virus
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43 envelope and play crucial roles in the virus interactions with the host cell (Cecchinato et al., 2010;

44 Naylor et al., 2010). The G protein, in particular, is vital for virus attachment to the cell membrane
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45 and contains major neutralizing epitopes. Its biological role, along with the viral high evolutionary

46 rate, contributes to the genetic heterogeneity of this region. As a result, the G protein region is often
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47 targeted for genotypic characterization, phylogenetic, and molecular epidemiological studies,

48 making it the most frequently sequenced region of the aMPV genome (Franzo et al., 2020).

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059
49 aMPV has long been acknowledged as responsible for production drop and economic losses within

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50 the global poultry industry, but its significance seems to have grown in recent years. aMPV subtype

51 A was first identified in South Africa in the 1970s and subsequently spread to several European

52 countries (Buys et al., 1989; Jones, 2010). Around the same time, another subtype, aMPV-B, was

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53 recognized and gradually became dominant in the Old World (Franzo et al., 2020; Salles et al.,

54 2023; Umar et al., 2016). The U.S. were unaffected until 1996, when aMPV subtype C emerged in

55 turkeys and wild birds (Seal, 2000; Toquin et al., 2006; Turpin et al., 2008). aMPV-C is

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56 predominantly established in North America and was thereafter sporadically detected in Europe and

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57 Asia (Graziosi et al., 2022b; Lee et al., 2007; Toquin et al., 2006; Tucciarone et al., 2022; van

58 Boheemen et al., 2012; Wei et al., 2013). However, North American and Eurasian AMPV-C strains

59
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belong to two separate lineages, with the former showing tropism primarily for Galliformes and the

60 latter for anatids (Brown et al., 2019). Another subtype, aMPV-D, was identified only in
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61 retrospective French samples from the 1980s (Bayon-Auboyer et al., 2000). Although two new

62 subtypes were detected in North America in black-backed gulls and monk parakeet chicks (Canuti

63 et al., 2019; Retallack et al., 2019), aMPV-C remained the principal subtype affecting U.S. flocks
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64 until very recently, when aMPV-B spread to the U.S. (Luqman et al., 2024). Additionally,

65 sequences of aMPV-A from strains collected in the U.S. have recently been published (GenBank
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66 Acc. Numbers: PP442011 and PP442012).

67 While focusing on the significant clinical and productive implications, the sources of aMPV
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68 introduction into North America have not been investigated yet. The generally limited and scattered

69 sampling and sequencing efforts impede the establishment of direct epidemiological connections on
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70 a global scale. However, some techniques based on molecular epidemiology and biostatistics allow

71 to partially compensate for this lack of information. To this purpose, the present study employs
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72 phylogenetic and phylogeographic approaches to estimate the source and the timing of introduction

73 of aMPV subtypes A and B into the U.S.

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059
74 All aMPV-A and -B sequences of the partial G gene for which the date and country of collection

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75 were known were downloaded from GenBank. After a quality check (i.e. absence of obvious

76 misalignment, unknown bases, premature stop-codons or frameshift mutations), only suitable

77 sequences were maintained in the dataset. Independent datasets were generated for the two aMPV

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78 subtypes. The analyzed region was selected to achieve the best compromise between sequence

79 length and country-year representativeness. After sequence alignment with MAFFT (Standley,

80 2013), a preliminary tree was reconstructed using IQ-Tree (Nguyen et al., 2015) selecting the

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81 substitution model with the lowest Bayesian Information Criteria (BIC) calculated using the same

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82 software. All strains clustering with vaccine ones were identified and removed from the study.

83 The datasets were analysed to reconstruct several population parameters, including time to the most
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84 recent common ancestor (tMRCA), evolutionary rate, and viral population dynamics using the

85 Bayesian serial coalescent approach implemented in BEAST 1.10 (Suchard et al., 2018). For each
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86 dataset, the nucleotide substitution model was selected based on the BIC score calculated using

87 JmodelTest (Darriba et al., 2012). The molecular clock was selected calculating the marginal

88 likelihood estimation through path-sampling and stepping-stone methods, as suggested by Baele et

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89 al. (Baele et al., 2012). The non-parametric Bayesian Skygrid (Hill and Baele, 2019) was selected to
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90 reconstruct viral population changes over time (relative genetic diversity: Effective population

91 size∙generation time; Ne∙τ). A discrete state phylogeographic analysis was also performed as

92 described by Lemey et al.(Lemey et al., 2009), implementing an asymmetric migration model with
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93 Bayesian stochastic search variable selection (BSSVS), allowing the identification of the most

94 parsimonious description of the spreading process and calculating a Bayesian Factor (BF) indicative
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95 of the statistical significance of the inferred migration path between geographic areas. Due to the

96 sparse nature of the sequence-country combination and the likely missing sampling in several
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97 countries, and in order to obtain a more balanced dataset, the analysis was also performed

98 aggregating countries in macro-areas considering their spatial proximity and geopolitical factors

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059
99 (i.e. Africa, Asia, Central America, Europe, Middle East, North America, and South America). Two

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100 independent runs of 200 million generations were performed. The log and tree files were merged

101 using logcombiner after the removal of a burn-in of 20%. Results were analysed using Tracer 1.7

102 and accepted only if the estimated sample size (ESS) was greater than 200 and the convergence and

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103 mixing were adequate. Parameter estimation was summarized in terms of mean and 95% highest

104 posterior density (HPD). Maximum clade credibility (MCC) trees were constructed and annotated

105 using TreeAnnotator (BEAST package). SpreaD3 (Bielejec et al., 2016) was used to calculate the

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106 BF associated with each migration route. All non-zero transition rates among countries were

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107 considered significant if the calculated BF was greater than 10. Additional summary statistics and

108 graphical outputs were generated using homemade R scripts (Team, 2014).

109
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The phylogeographic analysis of aMPV-B estimated that the North-American strains likely

110 originated from Eastern Asia, particularly Thailand. The time to the most recent common ancestor
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111 (tMRCA) of the U.S. clade was predicted around 2022 [95HPD:2021.6-2023.7] (Figure 1 and

112 Supplementary figure 1), which testifies to the relatively swift identification of the viral emergence.

113 The most closely related strains from Thailand were sampled in 2021, and the tMRCA of the
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114 overall clade was estimated in 2019 (Supplementary figure 1). The relatively long time elapsed—at
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115 least from an RNA virus evolution perspective—between the detection of these two groups

116 weakens the strength of inference regarding the introduction route. However, avian influenza virus

117 (AIV) migration from Eastern Asia to North America has been previously reported multiple times
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118 and has been mediated by migratory birds with overlapping flight paths (Bevins et al., 2016; Harvey

119 et al., 2023; Lee et al., 2015). The East Asian and Pacific American flight paths intersect in the
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120 regions of Eastern Russia and Alaska around the Bering Strait. It is estimated that about 1.5-3

121 million aquatic birds move from Asia to Alaska annually during the breeding season,, providing a
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122 significant potential for viral dispersal (Winker et al., 2007; Winker and Gibson, 2010).

123 Interestingly, connections between Eastern Europe and Asia, reportedly mediated by wild birds for

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124 AIV (Lee et al., 2015), have also been inferred in this study for aMPV-B, supporting the relevance

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125 of this spreading mechanism (Figure 2).

126 aMPV detections in wild birds, although limited and sporadic, have been reported for several aMPV

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127 subtypes, including aMPV-B (Canuti et al., 2019; Graziosi et al., 2022a; Heffels-Redmann et al.,

128 1998; Tucciarone et al., 2022; Turpin et al., 2008). Moreover, increasing evidence suggests that

129 some wild bird taxa could play a role in aMPV epidemiology. Particularly, wild ducks, geese, gulls,

130 and pheasants have proven to be susceptible to aMPV (Graziosi et al., 2022a). Due to their host

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131 ecology, these birds might act as viral carriers or reservoirs.

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132 Wild birds were already demonstrated to play a role in aMPV subtype C maintenance in the U.S.,

133 where the infection was serologically confirmed in American coots, American crows, Canada

134
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geese, cattle egrets, and rock pigeons (Turpin et al., 2008). aMPV-C was detected in geese,

135 sparrows, and starlings sampled in areas neighbouring turkey farms, confirming that wild birds
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136 could act as sources of infection for domestic turkeys (Shin et al., 2000). Waterfowl has been found

137 positive also in Canada (Jardine et al., 2018), widening the areas where wild birds can cross path
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138 over different flyways.

139 Although other pathways cannot be excluded, their likelihood seems lower. Being the U.S.
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140 primarily an exporter, the role of live animal importation should be discounted, and illegal

141 importation of poultry or poultry products into North America seems unlikely due to the difficulty
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142 of smuggling live birds or uncooked products with current transportation biosecurity measures (Lee

143 et al., 2015). The involvement of the Atlantic Rim in virus importation from Europe to North
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144 America through wild bird migration has recently been proposed for AIV (Caliendo et al., 2022).

145 However, the occurrence of such events seems lower, and this hypothesis is further questioned by

146 the mediation of Asian countries reported herein.

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147 Following its introduction in North America, other factors could have been involved in the

148 spreading process. This region is covered by different North-to-South flight paths through America
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149 (i.e. the Pacific, Central, Mississippi, and Atlantic flyways). The within flyways AIV transmission

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150 is well established, and inter-flyways viral passages can occur, albeit at a lower rate. Interestingly,

151 the Alaskan region is in connection with localities belonging to all American flyways, confirming

152 its relevance as a hub for wild bird (and associated virus) migration (Fourment et al., 2017).

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153 Such flyways might also have been involved in the introduction of aMPV-A from Mexico to the

154 U.S., as inferred by the performed phylogeographic analysis (Figure 1 and Supplementary figure 2).

155 Mexico is also on the route of all American flyways, which converge and overlap on this country

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156 (Cerda-Armijo et al., 2020). Unfortunately, the limited number of sequences and the long branch

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157 separating the Mexican-U.S. clade from the remaining strains prevent reliable conclusions. The

158 direct introduction of aMPV from Asia to Mexico through wild bird migration can be excluded.
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159 Potentially, aMPV-A might have followed the same pathway as aMPV-B, being introduced from

160 the Beringian crucible and then migrating southward without establishing in the U.S. After
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161 successfully infecting the Mexican poultry population, it might have spread northwards during

162 subsequent migratory seasons. However, the contribution of legal or illegal poultry trade or

163 importation cannot be excluded with confidence also in this case. Moreover, aMPV-A and B
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164 detections in wild birds from South America were reported (Felippe et al., 2011; Rizotto et al.,
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165 2019). Of note, a statistically significant migration route was inferred from Asia (South Korea) to

166 South America (Brazil), involving a lineage of European origin (Figure 1 and 2).
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167 Since the information on wild birds role in aMPV epidemiology is still scarce and scattered and

168 considering the significant practical implications for the poultry industry demonstrated by recent

169 U.S. outbreaks, further surveys of wild birds are encouraged. A better understanding of the
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170 poultry/wild bird interface in aMPV epidemiology and a more comprehensive characterization of its

171 host range extent are needed.

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172 References

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059
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286 97–103. https://doi.org/10.1007/S11262-005-5850-3

287 Tucciarone, C.M., Franzo, G., Legnardi, M., Pasotto, D., Lupini, C., Catelli, E., Quaglia, G., Graziosi, G., Dal
288 Molin, E., Gobbo, F., Cecchinato, M., 2022. Molecular Survey on A, B, C and New Avian
289 Metapneumovirus (aMPV) Subtypes in Wild Birds of Northern-Central Italy. Vet Sci 9.
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290 https://doi.org/10.3390/VETSCI9070373

291 Turpin, E.A., Stallknecht, D.E., Slemons, R.D., Zsak, L., Swayne, D.E., 2008. Evidence of avian
292 metapneumovirus subtype C infection of wild birds in Georgia, South Carolina, Arkansas and Ohio,
293 USA. Avian Pathol 37, 343–351. https://doi.org/10.1080/03079450802068566
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294 Umar, S., Sabir, H., Ahmed, A., Subhan, S., 2016. Avian metapneumovirus infection in poultry. Worlds Poult
295 Sci J 72, 833–846. https://doi.org/10.1017/S0043933916000738

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296 van Boheemen, S., Bestebroer, T.M., Verhagen, J.H., Osterhaus, A.D.M.E., Pas, S.D., Herfst, S., Fouchier,
297 R.A.M., 2012. A Family-Wide RT-PCR Assay for Detection of Paramyxoviruses and Application to a

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298 Large-Scale Surveillance Study. PLoS One 7, e34961. https://doi.org/10.1371/JOURNAL.PONE.0034961

299 Wei, L., Zhu, S., Yan, X., Wang, J., Zhang, C., Liu, S., She, R., Hu, F., Quan, R., Liu, J., 2013. Avian
300 metapneumovirus subgroup C infection in chickens, China. Emerg Infect Dis 19, 1092–1094.
301 https://doi.org/10.3201/eid1907.121126

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302 Winker, K., Gibson, D.D., 2010. The Asia-to-America Influx of Avian Influenza Wild Bird Hosts Is Large.
303 https://doi.org/10.1637/8741-032509-Reg.1 54, 477–482. https://doi.org/10.1637/8741-032509-
304 REG.1

305 Winker, K., McCracken, K.G., Gibson, D.D., Pruett, C.L., Meier, R., Huettmann, F., Wege, M., Kulikova, I. V.,
306 Zhuravlev, Y.N., Perdue, M.L., Spackman, E., Suarez, D.L., Swayne, D.E., 2007. Movements of Birds and

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307 Avian Influenza from Asia into Alaska - Volume 13, Number 4—April 2007 - Emerging Infectious
308 Diseases journal - CDC. Emerg Infect Dis 13, 547–552. https://doi.org/10.3201/EID1304.061072

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309

310 Captions er
311 Figure 1. Maximum clade credibility tree estimated based on the partial G-gene alignment of

312 aMPV-A (left) and aMPV-B (right). Branches have been color-coded based on the strain sampling
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313 regions and predicted ancestral locations. Further details, including strain name and location

314 posterior probability, are available in Supplementary Figures 1 and 2.

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315 Figure 2. aMPV-B genotype migration paths. Well supported migration paths (i.e. BF>21) among

316 areas are depicted. The arrows indicate the directionality of the process, while the edge colour is
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317 proportional to the base-10 logarithm of the Bayesian factor. The location of each area has been

318 matched with its centroid.

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319 Supplementary figure 1. Maximum clade credibility tree estimated based on the partial G-gene

320 alignment of aMPV-B. Branches have been color-coded based on the strain sampling regions and
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321 predicted ancestral locations, whose posterior probability is reported nearby the corresponding

322 node.
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323 Supplementary figure 2. Maximum clade credibility tree estimated based on the partial G-gene

324 alignment of aMPV-A. Branches have been color-coded based on the strain sampling regions and

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325 predicted ancestral locations, whose posterior probability is reported nearby the corresponding

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326 node.

327

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328 Founding

329 This work was supported by the Department of Animal Medicine, Production and Health,

330 University of Padua [grant number BIRD208917/20; “Project: Ricerca e tipizzazione di avian

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331 Metapneumovirus (aMPV) in volatili selvatici”] and EU funding within the NextGeneration EU-

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332 MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no.

333 PE00000007, INF-ACT).

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4811059