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\begin{document}

\title{Molecular identification and phylogenetic characterization of influenza
A virus at a wildlife-livestock interface in Mexico}



\author[1]{Jessica Mateus-Anzola}%
\author[1]{Liliana Gaytan-Cruz}%
\author[1]{Cecilia Montoya-Carrillo}%
\author[1]{Ivan Sanchez-Betancourt}%
\author[2]{Heliot Zarza}%
\author[3]{RENE Segura-Velázquez}%
\author[1]{Rafael Ojeda-Flores}%
\affil[1]{Universidad Nacional Autónoma de México}%
\affil[2]{Universidad Autónoma Metropolitana Unidad Lerma}%
\affil[3]{Universidad Nacional Autonoma de Mexico}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Influenza A virus (IAV) outbreaks constitute a constant threat to public
health and pose a remarkable impact on socio-economic systems worldwide.
Interactions between wild and domestic birds, humans, and swine can lead
to spillover events. Backyard livestock systems in proximity to wetlands
represent a high-risk area for viral spread. However, some gaps remain
in our knowledge of IAV transmission at the wildlife -- livestock
interface in Mexico. Hence, the study aimed at molecular identification
and phylogenetic characterization of IAV in the wild duck -- backyard
livestock interface at a wetland of Mexico. A total of 875 animals were
tested by real-time RT-PCR (qRT-PCR). We detected IAV in 3.68\% of the
wild ducks sampled during the winter season 2016 -- 2017. Nonetheless,
the samples obtained from backyard poultry and swine tested negative.
The highest IAV frequency (11.10\%) was found in the Mexican duck (Anas
diazi). Subtypes H1N1, H3N2, and H5N2 were detected. Phylogenetic
analysis of influenza viruses isolated from wild ducks of the Lerma
marshes revealed that hemagglutinin (HA) gene sequences were related to
waterfowl, swine, and poultry IAV strains previously isolated in the
United States and Mexico. In conclusion, the co-circulation of three IAV
subtypes in wild ducks close to backyard farms in Mexico, as well as,
the local identification of HA gene sequences genetically related to
Mexican livestock IAV strains and also to North American waterfowl IAV
strains, highlight the importance of the Lerma marshes for influenza
surveillance given the close interaction among wild birds, poultry,
pigs, and humans.%
\end{abstract}%



\sloppy


\textbf{Molecular identification and phylogenetic characterization of
influenza A virus at a wildlife-livestock interface in Mexico}

\textbf{Influenza virus at a wildlife-livestock interface}

Jessica Mateus-Anzola\textsuperscript{a}, Liliana
Gaytan-Cruz\textsuperscript{a}, Cecilia
Montoya-Carrillo\textsuperscript{a}, Jos\selectlanguage{ngerman}é Ivan
Sánchez-Betancourt\textsuperscript{b}, Heliot Zarza\textsuperscript{c},
René Segura-Velázquez\textsuperscript{d}, Rafael
Ojeda-Flores*\textsuperscript{a}.

\textsuperscript{a} Departamento de Etología, Fauna Silvestre y Animales
de Laboratorio. Facultad de Medicina Veterinaria y Zootecnia.
Universidad Nacional Autónoma de México. Ciudad de México, México.

\textsuperscript{b} Departamento de Medicina y Zootecnia de Cerdos.
Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional
Autónoma de México. Ciudad de México, México.

\textsuperscript{c} Departamento de Ciencias Ambientales, CBS.
Universidad Autónoma Metropolitana Unidad Lerma. Estado de México,
México.

\textsuperscript{d} Unidad de Investigación. Facultad de Medicina
Veterinaria y Zootecnia. Universidad Nacional Autónoma de México. Ciudad
de México, México.

\textbf{* Corresponding author: Rafael Ojeda Flores.} Laboratorio de
Ecología de Enfermedades y Una Salud. Facultad de Medicina Veterinaria y
Zootecnia. Universidad Nacional Autónoma de México. Av. Universidad
3000, Edificio A, Delegación Coyoacán, Col. Ciudad Universitaria, 04510
Ciudad de México, México.

Email address: ojedar@unam.mx

\textbf{Summary}

Influenza A virus (IAV) outbreaks constitute a constant threat to public
health and pose a remarkable impact on socio-economic systems worldwide.
Interactions between wild and domestic birds, humans, and swine can lead
to spillover events. Backyard livestock systems in proximity to wetlands
represent a high-risk area for viral spread. However, some gaps remain
in our knowledge of IAV transmission at the wildlife -- livestock
interface in Mexico. Hence, the study aimed at molecular identification
and phylogenetic characterization of IAV in the wild duck -- backyard
livestock interface at a wetland of Mexico. A total of 875 animals were
tested by real-time RT-PCR (qRT-PCR). We detected IAV in 3.68\% of the
wild ducks sampled during the winter season 2016 -- 2017. Nonetheless,
the samples obtained from backyard poultry and swine tested negative.
The highest IAV frequency (11.10\%) was found in the Mexican duck
(\emph{Anas diazi} ). Subtypes H1N1, H3N2, and H5N2 were detected.
Phylogenetic analysis of influenza viruses isolated from wild ducks of
the Lerma marshes revealed that hemagglutinin (HA) gene sequences were
related to waterfowl, swine, and poultry IAV strains previously isolated
in the United States and Mexico. In conclusion, the co-circulation of
three IAV subtypes in wild ducks close to backyard farms in Mexico, as
well as, the local identification of HA gene sequences genetically
related to Mexican livestock IAV strains and also to North American
waterfowl IAV strains, highlight the importance of the Lerma marshes for
influenza surveillance given the close interaction among wild birds,
poultry, pigs, and humans.

\textbf{Keywords:} backyard poultry, backyard swine, hunter-harvested
wild ducks, phylogenetic analysis, qRT-PCR, the Lerma marshes.

\textbf{Introduction}

Outbreaks of influenza A viruses (IAV) have caused considerable harm to
the animal and human health with several global socio-economic impacts
(McLeod, Morgan, Prakash, \& Hinrichs, 2005). Wild aquatic birds such as
the Anseriformes and Charadriiformes are the major natural reservoirs of
IAV, except H17 and H18 subtypes detected in bats (Munster et al., 2007;
Tong et al., 2013). In addition to its natural reservoir circulation,
IAV can cross the species barrier and infect a wide range of hosts
including, canids, equids, humans, poultry, and swine (Joseph, Su,
Vijaykrishna, \& Smith, 2017).

Wild waterfowl migration has a dominant role in the global influenza
virus spread (Franklin et al., 2019; Ren et al., 2016). Every year, low
pathogenic avian influenza (LPAI) outbreaks among waterfowl occur during
migration when contact rates among waterfowl populations are high (van
Dijk et al., 2014). Indeed, migratory wild birds are associated with
intra- and intercontinental dissemination of IAV as well as the
geographic dispersion of IAV subtypes in wild bird species and poultry
residents (Fourment, Darling, \& Holmes, 2017; Humphreys et al., 2020).

The wildlife -- livestock interface represents a significant source of
disease in domestic flocks (Wang, Jiang, Jin, Tan, \& Xu, 2013). Some
production practices such as free-range farming may increase the
likelihood of contact among wild and domestic populations, and represent
a critical factor for persistence and transmission of IAV (Cappelle et
al., 2014). Likewise, backyard systems are considered suitable areas for
influenza surveillance due to the complex interactions between swine,
poultry, humans, and wild animals (Hamilton-West et al., 2012;
Jimenez-Bluhm et al., 2018).

Previous research has provided some insights into the dynamic of IAV at
the wildlife --livestock interface (Wiethoelter, Beltrán-Alcrudo, Kock,
\& Mor, 2015). Phylogenetic analyses have revealed high sequence
homology among IAV circulating in pigs, poultry, and wild birds
(Bergervoet et al., 2019; Osbjer et al., 2016). Ecological and
phylogeographical analyses have shown that migratory waterfowl, domestic
ducks, free\selectlanguage{english}-ranging animals, and agroecosystems are crucial in the
maintenance and viral spread (Wei, Lin, \& Xie, 2015). Serological and
molecular studies of IAV have evidenced the viral circulation in
backyard farms situated close to ``el Yali'', one of the most important
wetlands in South America (Bravo-Vasquez et al., 2016).

The Lerma marshes constitute a critical surveillance area where wild
ducks, backyard poultry, backyard pigs, and humans co-occur in time and
space. Some molecular studies in wild ducks and their environment have
been conducted in this Natural Protected Area (Cuevas-Dominguez et al.,
2009; Ornelas-Eusebio, Obregon-Ascencio, Chavez-Maya, \&
Garcia-Espinosa, 2015; Ramirez-Martinez, Loza-Rubio, Mosqueda,
Gonzalez-Garay, \& Garcia-Espinosa, 2018). Nevertheless, the aims of
these studies were no centered on the interrelation among diverse
species, and a persistent gap in knowledge about IAV dynamics in the
wildlife -- livestock interface remains in the region. Therefore, this
study performed molecular and phylogenetic characterization of IAV in
wild ducks, backyard swine, and backyard poultry at this interface of
Central Mexico.

\textbf{Materials and methods}

\emph{Ethics Statement}

All procedures and protocols were approved by the ethical committee
``Subcomite Institucional para el Cuidado y Uso de Animales de
Experimentacion -- SICUAE'' (MC-2017/2-5, MC-2017/2-6), Universidad
Nacional Autonoma de Mexico (UNAM).

\emph{Study area}

This study was carried out in the Municipality of Lerma de Villada,
State of Mexico (19deg13'--19deg26'N, 99deg22'--99deg34'W). The Lerma marshes
provide a suitable habitat for thousands of migratory waterfowl than
arrived annually from Canada and the United States in the Mexican
Central Plateau on their migratory route to the south (Barragan Severo,
Lopez-Lopez, \& Babb Stanley, 2002). Animals were sampled from ten
different study units: wild ducks from the Lerma marshes, as well as
pigs and poultry from nine backyard farms located within a 6 Km radius
of the Lerma marshes boundaries (Figure 1).

\emph{Sample collection}

Wild ducks from the Lerma marshes and the surrounding backyard farms
were sampled during the winter and the early spring season 2016 -- 2017.
Each wild bird species was identified based on the descriptions provided
by Howell \& Webb (2010) and classified according to Clements et al.
(2019). The sample selection was done by a convenience sampling method.
Cloacal swabs from 597 hunter-harvested wild ducks, nasal swabs from 175
pigs, as well as cloacal and oropharyngeal swabs from 103 poultry (hens,
cocks, ducks, geese, and turkeys) were collected and stored in cryovials
containing 1 ml of Brain Heart Infusion (BHI) broth and 1ml of Eagle's
Minimum Essential Medium (EMEM) in wild duck/poultry and pig samples,
respectively. The biological samples were kept refrigerated at 4 degC
during transportation and stored at \selectlanguage{english}-80 degC until analysis.

\emph{qRT-PCR and viral isolation}

All samples were centrifugated at 2500 rpm for 5 minutes (mini
centrifuge with A12-2P rotor ``Dlab: D2012 plus''). The biological
samples were pooled by species and collection date (3 to 4
samples/pool). Subsequently, automated RNA extraction was performed from
200\selectlanguage{greek}μ\selectlanguage{english}L of each pooled supernatant using the Cador\selectlanguage{ngerman}® Pathogen 96 QIAcube HT
Kit (Qiagen) following the manufacturer's instructions. The qRT-PCR was
carried out with the VetMAX\selectlanguage{english}-Gold SIV Detection Kit (Life Technologies)
following the manufacturer's instructions (Ma et al., 2014). Negative
and positive controls were included in each run. Samples with a cycle
threshold value (Ct) \textless{}38 were considered positive as described
by the manufacturer. Positive pools were tested again individually.
Positive IAV samples were inoculated into the allantoic cavity of
9-to-11-day-old specific-pathogen-free (SPF) chicken embryos as
described before (Spackman \& Killian, 2014). The allantoic fluid was
harvested, and a haemagglutination assay was performed with 0.5\%
chicken red blood cells (Killian, 2014).

{\label{all-samples-were-centrifugated-at-2500-rpm-for-5-minutes-mini-centrifuge-with-a12-2p-rotor-dlab-d2012-plus.-the-biological-samples-were-pooled-by-species-and-collection-date-3-to-4-samplespool.-subsequently-automated-rna-extraction-was-performed-from-200ux3bcl-of-each-pooled-supernatant-using-the-cador-pathogen-96-qiacube-ht-kit-qiagen-following-the-manufacturers-instructions.-the-qrt-pcr-was-carried-out-with-the-vetmax-gold-siv-detection-kit-life-technologies-following-the-manufacturers-instructions-ma-et-al.-2014.-negative-and-positive-controls-were-included-in-each-run.-samples-with-a-cycle-threshold-value-ct-38-were-considered-positive-as-described-by-the-manufacturer.-positive-pools-were-tested-again-individually.-positive-iav-samples-were-inoculated-into-the-allantoic-cavity-of-9-to-11-day-old-specific-pathogen-free-spf-chicken-embryos-as-described-before-spackman-killian-2014.-the-allantoic-fluid-was-harvested-and-a-haemagglutination-assay-was-performed-with-0.5-chicken-red-blood-cells-killian-2014.}}

\emph{Sequencing}

Whole-genome sequencing was performed using an Ion Torrent Personal
Genome Machine (PGM) System (Life Technologies), following the
manufacturer's specifications. Sequencing libraries were constructed
using the Ion Plus Fragment Library (Life Technologies) by physical
fragmentation of genome segments with the Bioruptor(r) Sonication System
to generate fragments of approximately 200 base pairs (bp). The DNA
fragments were linked to Ion\selectlanguage{english}-compatible adapters and amplified using the
Ion PGM\selectlanguage{english}Hi\selectlanguage{english}-Q\selectlanguage{english} OT2 Kit (Life Technologies) on an Ion OneTouch\selectlanguage{english} 2 System.
The sequencing reaction was carried out using an Ion 314 Chip v2 and the
Ion PGM\selectlanguage{english} Hi\selectlanguage{english}-Q\selectlanguage{english} Sequencing Kit (Life Technologies). Reads were aligned
with a Q\selectlanguage{english}-score \selectlanguage{english}[?] 20 and assembled with the AssemblerSPAdes v 5.4.0
program. The average genomic coverage depth of the Ion Torrent PGM was
up to 300\selectlanguage{english}-fold.

\emph{Phylogenetic analysis}

The reference sequences used for the phylogenetic analysis were obtained
from the GenBank database (https://www.ncbi.nlm.nih.gov). The nucleotide
sequence identity selected was greater than 96\% using BLAST (Basic
Local Alignment Search Tool) from the National Center for
Biotechnology-NCBI (NCBI Resource Coordinators et al., 2018). Sequences
were aligned using the MUSCLE algorithm (Edgar, 2004). The phylogenetic
tree was constructed by the maximum-likelihood method with 1,000
bootstrap replicates in MEGA 7.0.26 (Kumar, Stecher, \& Tamura, 2016).

\textbf{Results}

\emph{AIV molecular detection}

A total of 875 biological samples of wild ducks (n=597), poultry
(n=103), and swine (n=175) were collected from November 2016 to May 2017
(Figure 2). Eight dabbling duck species and two diving duck species were
sampled for IAV detection by qRT-PCR in the Lerma marshes, State of
Mexico. Twenty-two wild dabbling ducks (3.68\%) tested positive for IAV
(Table 1). The IAV positive samples were obtained from November to
February, predominantly in the mid-winter season. The IAV positive wild
dabbling ducks belonged to four species: Green-winged Teal (\emph{Anas
crecca} ), Blue-winged Teal (\emph{Spatula discors} ), Northern Shoveler
(\emph{Spatula clypeata} ) and Mexican duck \emph{(Anas diazi} ) (Figure
3). Blue-winged Teal was the most frequent species in the sampling area,
followed by Green-winged Teal. The highest IAV frequency was found in
Mexican duck, followed by Northern Shoveler (Table 1). All swabs from
wild diving ducks, pigs, and poultry tested negative for IAV by qRT-PCR.

\emph{Phylogenetic characterization}

Nine IAV detected in wild dabbling ducks from the Lerma marshes were
isolated in chick embryos. All eight influenza gene segments of each
virus were sequenced, and the sequences were submitted into the GenBank
database (Table 2).

The phylogenetic analysis of the HA gene showed three major clades. The
upper clade had 3 strains
(A/blue-winged\_teal/EstadodeMexico/Lerma/UIFMVZ507/2017 (H5N2), A/
green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ80/2016 (H5N2), and
A/northern\_ shoveler/EstadodeMexico/Lerma/UIFMVZ613/2017 (H5N2))
related to Mexican swine and chicken sequences of H5N2 viral subtype
(Figure 4). Whereas one strain
(A/Mexican\_duck/EstadodeMexico/Lerma/UIFMVZ377/2016 (H5N2)) was
phylogenetically related to wild duck sequences of H5N1, H5N2, H5N5, and
H5N9 subtypes reported in United States of America (Figure 4). The HA
gene from A/blue-winged\_teal/EstadodeMexico/Lerma/UIFMVZ322/2016 (H1N1)
was found in a different clade, and it was strongly genetically related
to U.S. and Mexican swine H1N1 viruses (Figure 4). Finally, in the lower
clade were found four sequences (A/green-winged\_teal/
EstadodeMexico/Lerma/UIFMVZ169/2016 (H3N2),
A/green-winged\_teal/EstadodeMexico/ Lerma/UIFMVZ456/2017 (H3N2),
A/green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ 465/2017 (H3N2), and
A/blue-winged\_teal/EstadodeMexico/Lerma/UIFMVZ478/2017 (H3N2))
genetically similar to U.S. and Mexican swine H3N2 subtypes (Figure 4).

\textbf{Discussion}

This study describes the molecular identification and phylogenetic
characterization of IAV at a wildlife - livestock interface in the Lerma
marshes, Mexico. One of the findings in this study was the detection of
22 wild dabbling ducks (3.68\%) positive to IAV in the mid-winter.
Seasonality, geographical location, and between-year fluctuations can
modify the IAV prevalence and subtype distribution. For instance, higher
IAV prevalence (\textgreater{}30\%) occurs predominantly before and
during the fall migration, while lower prevalence (\textless{}1\%) is
related to the spring migration in North America (Lickfett, Clark,
Gehring, \& Alm, 2018; Wille, Brojer, Lundkvist, \& Jarhult, 2018).
However, the prevalence of AIVs in their natural hosts also depends on
the species (Munster et al., 2007).

Mallards (\emph{Anas platyrhynchos} ) and other dabbling duck species
(\emph{Anas} spp.) have higher IAV prevalence that diving ducks
(Vandegrift, Sokolow, Daszak, \& Kilpatrick, 2010). Specie-specific
differences in IAV prevalence are related to several factors including
intrinsic physiological characteristics, feeding behavior, shedding
patterns, population size, and adaptation to the virus (Munster et al.,
2007; Papp et al., 2017; J. G. van Dijk, Verhagen, Wille, \&
Waldenstrom, 2018; Wilcox et al., 2011). During 2016 - 2017, we detected
IAV in four wild dabbling duck species from the Lerma marshes: Mexican
Duck (11.10\%), Northern Shoveler (6.67\%), Green-winged Teal (4.97\%),
and Blue-winged Teal (4.28\%). These results are consistent with
multiyear surveillance conducted by Ferro et al. (2010), which found a
higher IAV prevalence in Blue-winged Teal, Northern Shoveler, and
Green-winged Teal in the United States. Likewise, in our study, none of
the diving duck species sampled resulted positive to IAV. This result
was in line with the research conducted by Munster et al. (2007), which
suggested less-efficient virus transmission by diving ducks.

Previous studies have identified IAV in waterfowl from the Lerma marshes
(Cuevas-Dominguez et al., 2009; Ramirez-Martinez et al., 2018). IAV
subtypes H7N3, H6N2, and H4N2 have been isolated from anatids, sentinel
ducks, and wild bird habitats of the State of Mexico (Barron-Rodriguez,
Chavez-Maya, Loza-Rubio, \& Garcia-Espinosa, 2018; Cuevas-Dominguez et
al., 2009; Ornelas-Eusebio et al., 2015). Findings in our study revealed
the co-circulation of three subtypes: H5N2, H3N2, and H1N1.

The phylogenetic analysis showed that the HA gene sequence of an H5N2
subtype isolated from a Mexican duck (\emph{A. diazi} ) was closely
related to migratory wild duck viruses from North America. This result
is consistent with the studies conducted by Barron-Rodriguez et al.
(2018) and Ornelas-Eusebio et al. (2015), who reported influenza viruses
highly similar to North America waterfowls IAV strains.

The phylogenetic characterization also revealed that the HA gene
sequences of the H5N2, H3N2, and H1N1 subtypes isolated from Northern
Shoveler, Green-winged Teal, and Blue-winged Teal shown to be more
phylogenetically related to swine and chicken sequences from Mexico. The
characterization of a swine-like or avian-like IAV isolated from wild
ducks is not very well documented. Nevertheless, Cuevas-Dominguez et al.
(2009) previously reported the isolation of an H7N3 subtype from a wild
bird that showed 100\% identity with an IAV isolated from a quail of the
United States. Similarly, Olsen, Karasin, and Erickson (2003) isolated
the H1N2 influenza virus (A/Duck/NorthCarolina/91347/01) in a wild duck
that was phylogenetically related to swine-like reassortant H1N2 and
H3N2 viruses of the United States of America.

Areas with close interactions between humans, livestock, and wild
animals have been considered as ``hotspots'' for IAV interspecies
transmission (Wei et al., 2015). Interspecies transmission of IAV is
dependent on several factors, including virus shedding, host immune
system, host receptor specificity, virus stability in the environment,
and the degree of close contact with different host species (Joseph et
al., 2017; Yassine, Lee, Gourapura, \& Saif, 2010). The presence of IAV
in wild migratory birds, along with the proximity of domestic flocks to
surface water has been significantly associated with increased risk of
local outbreaks (Karasin, Brown, Carman, \& Olsen, 2000; Walsh,
Amstislavski, Greene, \& Haseeb, 2017). However, in our study, all swine
and poultry samples were negative to IAV by qRT- PCR.

Negative IAV results could indicate a possible absence of recent viral
exposure at the time of sample collection or lack of viral adaptation to
alternate hosts such as poultry and swine (Awosanya, Babalobi, Omilabu,
Nguku, \& Ogundipe, 2013; Bourret et al., 2017). Likewise, some research
has reported zero prevalence in backyard systems during winter or
discordant detection of AIV subtypes in time and space between poultry
and wild birds (Jimenez-Bluhm et al., 2018; Verhagen et al., 2017).

In conclusion, this study identified the co-circulation of three
different subtypes of IAV in wild ducks close to backyard farms in the
Lerma marshes during the winter season 2016 -- 2017. Direct interspecies
transmission among wild and livestock animals was no determined.
Nevertheless, phylogenetic analysis suggests possible genetic
relationships among wild birds, swine, and poultry, as well as the
transboundary movement of influenza within the American continent most
likely associated with the waterfowl migration. Therefore, increased
long-term molecular IAV surveillance throughout the year in the Lerma
marshes would further contribute to understanding the contribution of
backyard animals, migratory, and resident wild ducks in the disease
epidemiology.

\textbf{Acknowledgements}

This research was funded by the PAPIIT-DGAPA projects Ndeg IA205916 and
IN222119. Jessica Mateus is a doctoral student of the `Posgrado en
Ciencias de la Produccion y de la Salud Animal', Universidad Nacional
Autonoma de Mexico (UNAM). The authors are grateful to the `Departamento
de Medicina y Zootecnia de Cerdos', and the `Unidad de Investigacion',
from the FMVZ\selectlanguage{english}-UNAM in particular to Rebeca Bautista Martinez and Brenda
Maya Badillo. We thank Gary Garcia Espinosa. We also extend our thanks
to the Mexican National Council for Science and Technology for
postgraduate fellowships (CONACyT, ID 785318).

\textbf{Conflict of Interest Statement}

The authors declare that they have no conflicts of interest.

\textbf{Data Availability Statement}

Data supporting the findings of this study are included in this article.
The genome sequences of influenza viruses are available in the NCBI
GenBank database.

\textbf{References}

Awosanya, E. J., Babalobi, O., Omilabu, S., Nguku, P., \& Ogundipe, G.
(2013). Prevalence and correlates of influenza-a in piggery workers and
pigs in two communities in Lagos, Nigeria. \emph{Pan African Medical
Journal} , \emph{16} . https://doi.org/10.11604/pamj.2013.16.102.1450

Barragan Severo, J., Lopez-Lopez, E., \& Babb Stanley, K. A. (2002).
Spatial and temporal variation patterns of a waterfowl community in a
reservoir system of the Central Plateau, Mexico. \emph{Hydrobiologia}
,\emph{467} (1/3), 123--131. https://doi.org/10.1023/A:1014969606303

Barron-Rodriguez, R. J., Chavez-Maya, F., Loza-Rubio, E., \&
Garcia-Espinosa, G. (2018). Isolation and characterization of influenza
A virus (H6N2) from a temporary artificial pond in
Mexico.\emph{Veterinaria Mexico OA} , \emph{5} (2).
https://doi.org/10.21753/5.2.475.

Bergervoet, S. A., Pritz-Verschuren, S. B. E., Gonzales, J. L., Bossers,
A., Poen, M. J., Dutta, J., \ldots{} Beerens, N. (2019). Circulation of
low pathogenic avian influenza (LPAI) viruses in wild birds and poultry
in the Netherlands, 2006--2016. \emph{Scientific Reports} , \emph{9}
(1), 13681. https://doi.org/10.1038/s41598-019-50170-8

Bourret, V., Lyall, J., Frost, S. D. W., Teillaud, A., Smith, C. A.,
Leclaire, S., \ldots{} Tiley, L. S. (2017). Adaptation of avian
influenza virus to a swine host. \emph{Virus Evolution} , \emph{3} (1).
https://doi.org/10.1093/ve/vex007

Bravo-Vasquez, N., Di Pillo, F., Lazo, A., Jimenez-Bluhm, P.,
Schultz-Cherry, S., \& Hamilton-West, C. (2016). Presence of influenza
viruses in backyard poultry and swine in El Yali wetland,
Chile.\emph{Preventive Veterinary Medicine} , \emph{134} , 211--215.
https://doi.org/10.1016/j.prevetmed.2016.10.004

Cappelle, J., Zhao, D., Gilbert, M., Nelson, M. I., Newman, S. H.,
Takekawa, J. Y., \ldots{} Xiao, X. (2014). Risks of Avian Influenza
Transmission in Areas of Intensive Free-Ranging Duck Production with
Wild Waterfowl. \emph{EcoHealth} , \emph{11} (1), 109--119.
https://doi.org/10.1007/s10393-014-0914-2

Clements J. F., T. S. Schulenberg, M. J. Iliff, S. M. Billerman, T. A.
Fredericks, B. L. Sullivan, \ldots{} C. L. Wood. (2019). \emph{The
eBird/Clements checklist of birds of the world: V2018.} (No. v2019).
Retrieved from The Cornell Lab of Ornithology website:
http://www.birds.cornell.edu/clementschecklist/download/

Cuevas-Dominguez, E., Gonzalez-Guzman, S., Quintana-Lopez, J. A.,
Loza-Rubio, E., Gonzalez-Rebeles, C., \& Garcia-Espinosa, G. (2009).
Deteccion de orthomyxovirus H7N3 en anatidos del Estado de
Mexico.\emph{Revista Electronica de Veterinaria} , \emph{10} .

Edgar, R. C. (2004). MUSCLE: a multiple sequence alignment method with
reduced time and space complexity. \emph{BMC Bioinformatics} ,\emph{5}
(1), 113. https://doi.org/10.1186/1471-2105-5-113

Ferro, P. J., Budke, C. M., Peterson, M. J., Cox, D., Roltsch, E.,
Merendino, T., \ldots{} Lupiani, B. (2010). Multiyear Surveillance for
Avian Influenza Virus in Waterfowl from Wintering Grounds, Texas Coast,
USA. \emph{Emerging Infectious Diseases} , \emph{16} (8), 1224--1230.
https://doi.org/10.3201/eid1608.091864

Fourment, M., Darling, A. E., \& Holmes, E. C. (2017). The impact of
migratory flyways on the spread of avian influenza virus in North
America. \emph{BMC Evolutionary Biology} , \emph{17} (1), 118.
https://doi.org/10.1186/s12862-017-0965-4

Franklin, Alan. B., Bevins, S. N., Ellis, J. W., Miller, R. S., Shriner,
S. A., Root, J. J., \ldots{} Deliberto, T. J. (2019). Predicting the
initial spread of novel Asian origin influenza A viruses in the
continental USA by wild waterfowl. \emph{Transboundary and Emerging
Diseases} , \emph{66} (2), 705--714. https://doi.org/10.1111/tbed.13070

Hamilton-West, C., Rojas, H., Pinto, J., Orozco, J., Herve-Claude, L.
P., \& Urcelay, S. (2012). Characterization of backyard poultry
production systems and disease risk in the central zone of
Chile.\emph{Research in Veterinary Science} , \emph{93} (1), 121--124.
https://doi.org/10.1016/j.rvsc.2011.06.015

Howell, S. N. G., \& Webb, S. (2010). \emph{A guide to the birds of
Mexico and northern Central America} (Repr). Oxford: Oxford Univ. Press.

Humphreys, J. M., Ramey, A. M., Douglas, D. C., Mullinax, J. M., Soos,
C., Link, P., \ldots{} Prosser, D. J. (2020). Waterfowl occurrence and
residence time as indicators of H5 and H7 avian influenza in North
American Poultry. \emph{Scientific Reports} , \emph{10} (1), 2592.
https://doi.org/10.1038/s41598-020-59077-1

Jimenez-Bluhm, P., Di Pillo, F., Bahl, J., Osorio, J., Schultz-Cherry,
S., \& Hamilton-West, C. (2018). Circulation of influenza in backyard
productive systems in central Chile and evidence of spillover from wild
birds. \emph{Preventive Veterinary Medicine} , \emph{153} , 1--6.
https://doi.org/10.1016/j.prevetmed.2018.02.018

Joseph, U., Su, Y. C. F., Vijaykrishna, D., \& Smith, G. J. D. (2017).
The ecology and adaptive evolution of influenza A interspecies
transmission. \emph{Influenza and Other Respiratory Viruses} ,\emph{11}
(1), 74--84. https://doi.org/10.1111/irv.12412

Karasin, A. I., Brown, I. H., Carman, S., \& Olsen, C. W. (2000).
Isolation and Characterization of H4N6 Avian Influenza Viruses from Pigs
with Pneumonia in Canada. \emph{Journal of Virology} , \emph{74} (19),
9322--9327. https://doi.org/10.1128/JVI.74.19.9322-9327.2000

Killian, M. L. (2014). Hemagglutination Assay for Influenza Virus. In E.
Spackman (Ed.), \emph{Animal Influenza Virus} (Vol. 1161, pp. 3--9).
https://doi.org/10.1007/978-1-4939-0758-8\_1

Kumar, S., Stecher, G., \& Tamura, K. (2016). MEGA7: Molecular
Evolutionary Genetics Analysis Version 7.0 for Bigger
Datasets.\emph{Molecular Biology and Evolution} , \emph{33} (7),
1870--1874. https://doi.org/10.1093/molbev/msw054

Lickfett, T. M., Clark, E., Gehring, T. M., \& Alm, E. W. (2018).
Detection of Influenza A viruses at migratory bird stopover sites in
Michigan, USA. \emph{Infection Ecology \& Epidemiology} , \emph{8} (1),
1474709. https://doi.org/10.1080/20008686.2018.1474709

Ma, M.-J., Yang, X.-X., Xia, X., Anderson, B. D., Heil, G. L., Qian,
Y.-H., \ldots{} Gray, G. C. (2014). Comparison of commercial influenza A
virus assays in detecting avian influenza H7N9 among poultry cloacal
swabs, China. \emph{Journal of Clinical Virology} , \emph{59} (4),
242--245. https://doi.org/10.1016/j.jcv.2014.01.009

McLeod, A., Morgan, N., Prakash, A., \& Hinrichs, J.
(2005).\emph{Economic and social impacts of avian influenza} . Retrieved
from
http://www.fao.org/avianflu/documents/Economic-and-social-impacts-of-avian-influenza-Geneva.pdf

Munster, V. J., Baas, C., Lexmond, P., Waldenstrom, J., Wallensten, A.,
Fransson, T., \ldots{} Fouchier, R. A. M. (2007). Spatial, Temporal, and
Species Variation in Prevalence of Influenza A Viruses in Wild Migratory
Birds. \emph{PLoS Pathogens} , \emph{3} (5), e61.
https://doi.org/10.1371/journal.ppat.0030061

NCBI Resource Coordinators, Agarwala, R., Barrett, T., Beck, J., Benson,
D. A., Bollin, C., \ldots{} Zbicz, K. (2018). Database resources of the
National Center for Biotechnology Information. \emph{Nucleic Acids
Research} , \emph{46} (D1), D8--D13. https://doi.org/10.1093/nar/gkx1095

Olsen, C. W., Karasin, A., \& Erickson, G. (2003). Characterization of a
swine-like reassortant H1N2 influenza virus isolated from a wild duck in
the United States. \emph{Virus Research} , \emph{93} (1), 115--121.
https://doi.org/10.1016/S0168-1702(03)00073-X

Ornelas-Eusebio, E., Obregon-Ascencio, A., Chavez-Maya, F., \&
Garcia-Espinosa, G. (2015). Molecular characterization of an influenza A
virus (H4N2) isolated from waterfowl habitats in the State of
Mexico.\emph{Journal of Veterinary Medical Science} , \emph{77} (3),
365--369. https://doi.org/10.1292/jvms.14-0267

Osbjer, K., Berg, M., Sokerya, S., Chheng, K., San, S., Davun, H.,
\ldots{} Zohari, S. (2016). Influenza A Virus in Backyard Pigs and
Poultry in Rural Cambodia. \emph{Transboundary and Emerging Diseases} .
https://doi.org/10.1111/tbed.12547

Papp, Z., Clark, R. G., Parmley, E. J., Leighton, F. A., Waldner, C., \&
Soos, C. (2017). The ecology of avian influenza viruses in wild dabbling
ducks (Anas spp.) in Canada. \emph{PLOS ONE} , \emph{12} (5), e0176297.
https://doi.org/10.1371/journal.pone.0176297

Ramirez-Martinez, L. A., Loza-Rubio, E., Mosqueda, J., Gonzalez-Garay,
M. L., \& Garcia-Espinosa, G. (2018). Fecal virome composition of
migratory wild duck species. \emph{PLOS ONE} , \emph{13} (11), e0206970.
https://doi.org/10.1371/journal.pone.0206970

Ren, H., Jin, Y., Hu, M., Zhou, J., Song, T., Huang, Z., \ldots{} Liang,
L. (2016). Ecological dynamics of influenza A viruses: Cross-species
transmission and global migration. \emph{Scientific Reports} ,\emph{6}
(1). https://doi.org/10.1038/srep36839

Spackman, E., \& Killian, M. L. (2014). Avian Influenza Virus Isolation,
Propagation, and Titration in Embryonated Chicken Eggs. In E. Spackman
(Ed.), \emph{Animal Influenza Virus} (Vol. 1161, pp. 125--140).
https://doi.org/10.1007/978-1-4939-0758-8\_12

Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M., \ldots{}
Donis, R. O. (2013). New World Bats Harbor Diverse Influenza A
Viruses.\emph{PLoS Pathogens} , \emph{9} (10), e1003657.
https://doi.org/10.1371/journal.ppat.1003657

van Dijk, J. G. B., Hoye, B. J., Verhagen, J. H., Nolet, B. A.,
Fouchier, R. A. M., \& Klaassen, M. (2014). Juveniles and migrants as
drivers for seasonal epizootics of avian influenza virus. \emph{Journal
of Animal Ecology} , \emph{83} (1), 266--275.
https://doi.org/10.1111/1365-2656.12131

van Dijk, J. G., Verhagen, J. H., Wille, M., \& Waldenstrom, J. (2018).
Host and virus ecology as determinants of influenza A virus transmission
in wild birds. \emph{Current Opinion in Virology} , \emph{28} , 26--36.
https://doi.org/10.1016/j.coviro.2017.10.006

Vandegrift, K. J., Sokolow, S. H., Daszak, P., \& Kilpatrick, A. M.
(2010). Ecology of avian influenza viruses in a changing world: Ecology
of avian influenza viruses in a changing world. \emph{Annals of the New
York Academy of Sciences} , \emph{1195} (1), 113--128.
https://doi.org/10.1111/j.1749-6632.2010.05451.x

Verhagen, J. H., Lexmond, P., Vuong, O., Schutten, M., Guldemeester, J.,
Osterhaus, A. D. M. E., \ldots{} Fouchier, R. A. M. (2017). Discordant
detection of avian influenza virus subtypes in time and space between
poultry and wild birds; Towards improvement of surveillance
programs.\emph{PLOS ONE} , \emph{12} (3), e0173470.
https://doi.org/10.1371/journal.pone.0173470

Walsh, M. G., Amstislavski, P., Greene, A., \& Haseeb, M. A. (2017). The
Landscape Epidemiology of Seasonal Clustering of Highly Pathogenic Avian
Influenza (H5N1) in Domestic Poultry in Africa, Europe and
Asia.\emph{Transboundary and Emerging Diseases} , \emph{64} (5),
1465--1478. https://doi.org/10.1111/tbed.12537

Wang, Y., Jiang, Z., Jin, Z., Tan, H., \& Xu, B. (2013). Risk Factors
for Infectious Diseases in Backyard Poultry Farms in the Poyang Lake
Area, China. \emph{PLoS ONE} , \emph{8} (6), e67366.
https://doi.org/10.1371/journal.pone.0067366

Wei, K., Lin, Y., \& Xie, D. (2015). Evolutionary and Ecological
Dynamics of Transboundary Disease Caused by H5N1 Virus in Southeast
Asia. \emph{Transboundary and Emerging Diseases} , \emph{62} (3),
315--327. https://doi.org/v

Wiethoelter, A. K., Beltran-Alcrudo, D., Kock, R., \& Mor, S. M. (2015).
Global trends in infectious diseases at the wildlife--livestock
interface. \emph{Proceedings of the National Academy of Sciences}
,\emph{112} (31), 9662--9667. https://doi.org/10.1073/pnas.1422741112

Wilcox, B. R., Knutsen, G. A., Berdeen, J., Goekjian, V., Poulson, R.,
Goyal, S., \ldots{} Stallknecht, D. E. (2011). Influenza-A Viruses in
Ducks in Northwestern Minnesota: Fine Scale Spatial and Temporal
Variation in Prevalence and Subtype Diversity. \emph{PLoS ONE} ,\emph{6}
(9), e24010. https://doi.org/10.1371/journal.pone.0024010

Wille, M., Brojer, C., Lundkvist, A., \& Jarhult, J. D. (2018).
Alternate routes of influenza A virus infection in Mallard (Anas
platyrhynchos). \emph{Veterinary Research} , \emph{49} (1), 110.
https://doi.org/10.1186/s13567-018-0604-0

Yassine, H. M., Lee, C.-W., Gourapura, R., \& Saif, Y. M. (2010).
Interspecies and intraspecies transmission of influenza A viruses:
Viral, host and environmental factors. \emph{Animal Health Research
Reviews} , \emph{11} (01), 53--72.
https://doi.org/10.1017/S1466252310000137

\textbf{Table 1.} Influenza A virus frequency in wild ducks from the
Lerma marshes, 2016 -- 2017.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \textbf{Scientific name} & \textbf{Common name} & \textbf{n} &
\textbf{IAV (\%)}+\tabularnewline
\midrule
\endhead
\textbf{Dabbling ducks} & \emph{Anas acuta} & Northern Pintail & 43 & 0
(0)\tabularnewline
& \emph{Anas crecca} & Green-winged Teal & 201 & 10
(4.97)\tabularnewline
& \emph{Anas diazi} & Mexican duck & 9 & 1 (11.1)\tabularnewline
& \emph{Mareca americana} & American Wigeon & 41 & 0 (0)\tabularnewline
& \emph{Mareca strepera} & Gadwall & 47 & 0 (0)\tabularnewline
& \emph{Spatula discors} & Blue-winged Teal & 210 & 9
(4.28)\tabularnewline
& \emph{Spatula clypeata} & Northern Shoveler & 30 & 2
(6.67)\tabularnewline
& \emph{Spatula cyanoptera} & Cinnamon Teal & 10 & 0 (0)\tabularnewline
\textbf{Diving ducks} & \emph{Aythya collaris} & Ring-necked duck & 3 &
0 (0)\tabularnewline
& \emph{Oxyura jamaicensis} & Ruddy duck & 3 & 0 (0)\tabularnewline
& \textbf{Total} & & \textbf{597} & \textbf{22 (3.68)}\tabularnewline
\bottomrule
\end{longtable}

+ by the qRT-PCR assay. Abbreviation used: IAV, Influenza A virus.

\textbf{Table 2.} NCBI GenBank accession numbers of Influenza A viruses
detected in wild ducks from the Lerma marshes.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
Influenza A virus+\strut
\end{minipage} & \begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
GenBank accession number\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ80/2016 (H5N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT325963-MT325970\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Mexican\_duck/EstadodeMexico/Lerma/UIFMVZ377/2016 (H5N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MK828137- MK828144\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Blue-winged\_teal/EstadodeMexico/Lerma/UIFMVZ507/2017 (H5N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT305332-MT305339\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Northern\_shoveler/EstadodeMexico/Lerma/UIFMVZ613/2017 (H5N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT325927-MT325934\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Blue-winged\_teal/EstadodeMexico/Lerma/UIFMVZ322/2016 (H1N1)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT311129-MT311136\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ169/2016 (H3N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT318090-MT318097\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ456/2017 (H3N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT318132-MT318139\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
A/Green-winged\_teal/EstadodeMexico/Lerma/UIFMVZ465/2017 (H3N2)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
MT318432-MT318439\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
A/Blue\_winged\_teal/EstadodeMexico/Lerma/UIFMVZ478/2017 (H3N2)\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
MT320087-MT320094\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

+ The nine IAV isolated in 9-to-11-day-old specific-pathogen-free (SPF)
chicken embryos.

\textbf{Figure 1.} Geographical distribution of the backyard farms in
Lerma, State of Mexico (n=9). Yellow circles indicate the poultry farms,
red circles show poultry and pig farms, the purple circle indicates a
pig farm, and the blue line indicates the Lerma marshes boundaries.

\textbf{Figure 2.} Timeline of sample collection in wild ducks, backyard
poultry, and backyard pigs. From Lerma, State of Mexico. The black
numbers represent the total of sampled animals. The circled numbers
indicate the ID numbers of the farms.

\textbf{Figure 3.} Influenza A virus frequency in wild dabbling ducks.
The relative percentage of wild ducks positive to IAV by specie and
sampling month.

\textbf{Figure 4.} Maximum-likelihood phylogenetic tree of the
hemagglutinin (HA) gene segment of Influenza A viruses from wild ducks
of Lerma, State of Mexico 2016 -- 2017. Mexican viruses isolated are
shown in colors by subtype: blue (H5N2), purple (H1N1), and red (H3N2).
Scale bars indicate nucleotide substitutions per site.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}

\selectlanguage{english}
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