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\begin{document}

\title{Avian influenza virus in water: global subtype diversity and spatial
distribution patterns}



\author[1]{Jessica Mateus-Anzola}%
\author[2]{Beatriz Martínez-López}%
\author[3]{Ana Cecilia Espinosa-García}%
\author[1]{Rafael Ojeda-Flores}%
\affil[1]{Universidad Nacional Autonoma de Mexico Facultad de Medicina Veterinaria y Zootecnia}%
\affil[2]{University of California Davis Department of Medicine and Epidemiology}%
\affil[3]{Universidad Nacional Autonoma de Mexico Instituto de Ecologia}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The current COVID-19 pandemic highlights the need for zoonotic
infectious disease surveillance. Avian influenza virus (AIV) poses a
significant threat to animal and public health due to its pandemic
potential. Virus-contaminated water has been suggested as an important
AIV spread mechanism among multiple species. Nevertheless, few studies
have characterized the global AIV subtype diversity and distribution in
environmental water. Therefore, this study aims to provide an updated
descriptive and phylogenetic analysis of AIVs isolated in water samples
from high risk-sites for influenza outbreaks (i.e., live bird markets,
poultry farms, and wild bird habitats) on a global scale. A total of 234
hemagglutinin (HA) gene sequences of 21 subtypes were reported from nine
countries between 2003 -- 2020. Fourteen AIV subtypes were solely
reported from Asian countries. Most of the viral sequences were obtained
in China and Bangladesh with 47.44\% and 23.93\%, respectively.
Likewise, the greatest global AIV subtype diversity was observed in
China with twelve subtypes. Live bird markets represented the main
sampling site for AIV detection in water samples (64.10\%), mostly from
poultry cage water. Nevertheless, the highest subtype diversity was
observed in water samples from wild bird habitats, especially from the
Izumi plain and the Dongting Lake located in Japan and China,
respectively. Water from drinking poultry troughs evidenced the greatest
subtype diversity in live bird markets, meanwhile, environmental water
used by ducks had the highest number of different subtypes in poultry
farms. The maximum-likelihood phylogenetic tree showed that some HA
sequences were closely related among different poultry/wild bird-related
environments from different geographic origins. Therefore, the results
suggest that even though the availability of HA gene sequences in
public-access databases varies greatly among countries, environmental
AIV surveillance represents a useful tool to elucidate potential viral
diversity in wild and domestic bird populations.%
\end{abstract}%



\sloppy


\textbf{Avian influenza virus in water: global subtype diversity and
spatial distribution patterns}

Jessica Mateus-Anzola \textsuperscript{a}, Beatriz
Mart\selectlanguage{ngerman}ínez-López\textsuperscript{b}, Ana Cecilia Espinosa-García
\textsuperscript{c}, Rafael Ojeda-Flores \textsuperscript{a*}

\textsuperscript{a} Laboratorio de Ecología de Enfermedades y Una Salud,
Departamento de Etología, Fauna Silvestre y Animales de Laboratorio,
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional
Autónoma de México, 04510, Mexico.

\textsuperscript{b} Center for Animal Disease Modeling and Surveillance
(CADMS), Department of Medicine \& Epidemiology, School of Veterinary
Medicine, University of California-Davis, Davis, CA, 95616, United
States.

\textsuperscript{c} Laboratorio Nacional de Ciencias de la
Sostenibilidad, Instituto de Ecología, Universidad Nacional Autónoma de
México, 04510, Mexico.

\selectlanguage{english}*\textbf{Corresponding author: Rafael Ojeda-Flores.} Laboratorio de
Ecologia de Enfermedades y Una Salud, Departamento de Etologia, Fauna
Silvestre y Animales de Laboratorio, Facultad de Medicina Veterinaria y
Zootecnia, Universidad Nacional Autonoma de Mexico, Av. Universidad
3000, Edificio A, Delegacion Coyoacan, Col. Ciudad Universitaria, 04510,
Mexico city, Mexico. Tel.: +52 5513207039.

\emph{E-mail address} : ojedar@unam.mx

\textbf{Summary}

The current COVID-19 pandemic highlights the need for zoonotic
infectious disease surveillance. Avian influenza virus (AIV) poses a
significant threat to animal and public health due to its pandemic
potential. Virus-contaminated water has been suggested as an important
AIV spread mechanism among multiple species. Nevertheless, few studies
have characterized the global AIV subtype diversity and distribution in
environmental water. Therefore, this study aims to provide an updated
descriptive and phylogenetic analysis of AIVs isolated in water samples
from high risk-sites for influenza outbreaks (i.e., live bird markets,
poultry farms, and wild bird habitats) on a global scale. A total of 234
hemagglutinin (HA) gene sequences of 21 subtypes were reported from nine
countries between 2003 -- 2020. Fourteen AIV subtypes were solely
reported from Asian countries. Most of the viral sequences were obtained
in China and Bangladesh with 47.44\% and 23.93\%, respectively.
Likewise, the greatest global AIV subtype diversity was observed in
China with twelve subtypes. Live bird markets represented the main
sampling site for AIV detection in water samples (64.10\%), mostly from
poultry cage water. Nevertheless, the highest subtype diversity was
observed in water samples from wild bird habitats, especially from the
Izumi plain and the Dongting Lake located in Japan and China,
respectively. Water from drinking poultry troughs evidenced the greatest
subtype diversity in live bird markets, meanwhile, environmental water
used by ducks had the highest number of different subtypes in poultry
farms. The maximum-likelihood phylogenetic tree showed that some HA
sequences were closely related among different poultry/wild bird-related
environments from different geographic origins. Therefore, the results
suggest that even though the availability of HA gene sequences in
public-access databases varies greatly among countries, environmental
AIV surveillance represents a useful tool to elucidate potential viral
diversity in wild and domestic bird populations.

\textbf{Keywords:} Environmental Monitoring; Epidemiology;
Hemagglutinin; Infectious disease; Phylogenetic Analysis; Water
Microbiology.

\textbf{Introduction}

Waterborne transmission is considered a significant route for avian
influenza viruses (AIV) dissemination (Lebarbenchon et al., 2011). This
indirect route of transmission is known as one of the main determinants
of disease dynamics, since water can be simultaneously contaminated by
different viral strains and infect multiple animals, including wild
birds (Numberger et al., 2019; Roche et al., 2009).

Waterfowl are recognized as the natural reservoir of IAV and play a key
role in the viral spread and diversity (Montalvo-Corral \& Hernandez,
2010). Infected wild ducks shed feces with high AIV concentrations into
aquatic environments (Dovas et al., 2010). Viruses shedding into the
water have the potential to infect any other waterfowl within bird
populations by drinking or filtering the virus-contaminated water
(Keeler et al., 2014). Therefore, viral transmission among wild aquatic
birds mainly occurs via the fecal-oral route through contaminated water
(Brown et al., 2007).

Environmental water where resident and migratory waterfowl congregate
has a significant role in the AIV ecology and epidemiology (Numberger et
al., 2019). Viruses shed before migration can persist in the water over
winter and become a source of infection to birds returning during spring
(Brown et al., 2007). This environmental persistence may enable short-
and long-term maintenance of AIV among spatially or temporally separated
wild bird populations (Lickfett et al., 2018).

Wild bird habitats close to livestock production systems also represent
high-risk zones for AIV interspecies transmission (Achenbach \& Bowen,
2011; Harris et al., 2017). Barnyard fowls from backyard farms can use
open waters and become indirectly infected by wild waterfowls
congregated in the same habitats (Cappelle et al., 2011). Likewise,
other farm animals, such as pigs, can be AIV infected by contaminated
surface water collected from natural or artificial ponds and used for
cleaning barns and watering animals (Karasin et al., 2000; Ma et al.,
2007).

In addition to the wild duck-livestock animal interface, live bird
markets constitute high-risk sites for harboring and spreading AIV (Khan
et al., 2018). Wet markets promote close contact between different
animal species which facilitates the genetic reassortment of influenza
viruses (Vergne et al., 2019). Wild and domestic birds (including
infected birds) use to share cages, feed, and water trays. Therefore,
birds from live bird markets can be co-infected with different AIV
subtypes (Khan et al., 2018).

Globally, multiple subtypes of low- and high-pathogenic avian influenza
(LPAI and HPAI) have been identified based on epidemiological
surveillance (Machalaba et al., 2015). Environmental AIV surveillance is
considered a convenient, noninvasive, and sensitive method for
conducting AIV detection (Leung et al., 2007). Avian influenza
surveillance in poultry-related environments, including live poultry
markets, wild bird habitats, slaughterhouses, and poultry farms is
conducted annually in some countries (Zhang et al., 2019). However, the
global diversity and distribution of AIVs in environmental water widely
used by waterfowl and poultry have been scarcely studied. In this study,
we aim to address this knowledge gap by characterizing the AIV diversity
and its spatial distribution in water samples from areas considered to
be at high risk for avian influenza outbreaks (i.e., wild bird habitats,
live bird markets, poultry farms) on a global scale.

\textbf{Materials and Methods}

\emph{Data collection from open-access databases}

A total of 2 920 AIV hemagglutinin (HA) gene sequences were collected
until 04 February 2021 using the keywords: ``environment'', ``water'',
``sewage'', ``wastewater'', ``surface\_water'', and ``drinking\_water''
from the NCBI Influenza Virus Database
(\emph{https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi}),
the GISAID EpiFlu\selectlanguage{english} Influenza database (\emph{https://www.gisaid.org/}),
the Influenza Research Database (IRD,\emph{https://www.fludb.org/}) and
the OpenFlu database (\emph{http://openflu.vital-it.ch/}). All non-water
sample sequences detected in animal environments and incomplete subtype
data were removed. The resulting dataset was de-replicated to remove
identical records based on the Accession Numbers (Table S1). The
remaining 234 HA gene sequences compose the final dataset used for the
data analysis.

\emph{Data analysis}

Data from subtype, country, sampling site, and water source were
extracted and analyzed. Sample sites were live bird market (LBM),
poultry farm (PF), wild bird habitat (WBH), or unknown (UNK). Water
sources were drinking water trough (DW), poultry cage water (PCW),
sewage (SW), turkey barn water (TBW), hand-wash water (HWW),
carcass-wash water (CWW), duck's environmental water (DEW), or unknown
(UNK). The category ``unknown'' referred to not clearly stated
information. Data analysis was conducted using graphs and maps created
in RStudio using the ggplot2 package (Wickham, 2016) and Mapcharnet(r)
(\emph{https://mapchart.net}).

\emph{Phylogenetic analysis}

From the 234 sequences dataset, 26 HA partial or identical sequences
were removed. A total of 208 complete HA gene sequences were aligned
using the MUSCLE algorithm (Edgar, 2004). A circular phylogenetic tree
was constructed by the Maximum-likelihood method with 1000 bootstrap
replicates using the best-fit nucleotide substitution model in MEGA X
(Kumar et al., 2018) and was edited with FigTree v1.4.4 software
(Rambaut, 2018). The nucleotide substitution model was selected using
the Akaike and Bayesian information criteria (AIC and BIC) (Kumar et
al., 2018).

\textbf{Results}

A total of 234 HA gene sequences of AIVs gathered in water samples were
reported from nine countries between 2003 -- 2020. No AIV sequences were
reported from Africa, Oceania, or South America. Most of the AIV
sequences were obtained from China (47.44\%, n=111) and Bangladesh
(23.93\%, n=56), mainly from two subtypes: LPAI H9N2 and HPAI H5N1. The
H10N7 was the only subtype described in Europe. Most subtypes (H3N2,
H4N8, H4N9, H5N1, H5N6, H5N8, H6N2, H6N6, H7N9, H9N2, H10N8, H11N2,
H11N9, and H12N7) were reported only by Asian countries.

The highest AIV diversity was observed in China with twelve subtypes
followed by Japan with six subtypes. H3N8 and H4N6 subtypes were
described in both Asian and American countries. H4N9, H7N9, H10N8,
H11N2, and H12N7 subtypes were exclusively reported in China. Similarly,
H4N2, H4N8, and H6N6 subtypes were solely detected in Mexico, Japan, and
Vietnam, respectively. Likewise, H1N1, H5N2, and H7N2 subtypes were only
isolated in the United States (Figure 1).

The AIV sequences were detected in water samples from live bird markets
(64.10\%, n=150), wild bird habitats (19.23\%, n=45), poultry farms
(12.82\%, n=30), and unknown sampling site (3.85\%, n=9). AIV sequences
from live bird markets were mainly reported by Asian countries (98.67\%,
n=148), especially China (54.66\%, n=82) and Bangladesh (37.33\%, n=56).
The United States was the only country on the American continent that
reported AIV sequences from a live bird market (1.33\%, n=2) (Figure
2A).

Likewise, AIV sequences from wild bird habitats and poultry farms were
detected in both Asia and America. Most of the AIV sequences from wild
bird habitats were reported in China (46.67\%, n=21) and Japan (35.56\%,
n=16) (Figure 2B), especially from the Dongting Lake (Hunan) and the
Izumi Plain (Kagoshima), respectively. However, IAV sequences were also
identified in water samples from the Qinghai lake in China and from a
Kodai pond in a Japanese zoo. In the American continent, the IAV
sequences were reported in Mexico, the United States, and Canada (Figure
2B). In particular, IAV sequences were detected from the Agassiz
National Wildlife Refuge and the Thief Lake Wildlife Management Area in
Minnesota, United States, filtered wetland water in Alaska, United
States, a wetland in Central Mexico, and a wild bird habitat in Alberta,
Canada.

The IAV sequences from poultry farms were mainly reported in the United
States (66.67\%, n=20) (Figure 2C), corresponding to water samples from
barns of turkey flocks in Minnesota and Nebraska. One IAV sequence
belonged to a free-range farm in the Netherlands. All the IAV sequences
(H3N2, H3N8, H4N9, H11N2, and H12N7) detected from poultry farms in
China (20\%, n=6) were located close to aquatic environments (i.e., a
domestic duck and goose farm close to a river in Hunan; as well as duck
farms in the Dongting Lake region, China). On the other hand, nine AIV
sequences (H5N1, H5N6, H6N6, H7N9, and H11N9) from four Asian countries
did not specify the sampling site (Figure 2D).

The highest subtype diversity was found in water samples from wild bird
habitats with eleven subtypes (H1N1, H3N8, H4N2, H4N6, H4N8, H5N1, H5H6,
H5N8, H6N2, H9N2, and H10N8). The wild bird habitats in Japan
represented the highest subtype diversity with six subtypes (H3N8, H4N6,
H4N8, H5N6, H5N8, and H6N2), two of them have also been reported from
North American wild bird habitats (H3N8 and H4N6) (Figure 2B).

In the context of live bird markets, seven subtypes (H3N2, H5N1, H5N6,
H6N2, H7N9, H9N2, and H11N9) were reported in Southeast Asia, and one
subtype (H7N2) was detected in the United States (Figure 2A). Similarly,
in poultry farms, six subtypes (H3N2, H3N8, H4N9, H5N1, H11N2, and
H12N7) were reported in Southeast Asia, one subtype (H10N7) in the
Netherlands, and one subtype (H5N2) in the United States (Figure 2C).
China evidenced the highest subtype diversity in both live bird markets
and poultry farms.

The live bird market samples were obtained from poultry cage water
(27.33\%, n=41), sewage (21.33\%, n=32), and contaminated drinking water
troughs (14.67\%, n=22). The greatest subtype diversity was found in
water from drinking troughs with six subtypes (H3N2, H5N1, H5N6, H6N2,
H9N2, and H11N9). However, a high percentage of the live bird market
samples did not report the water source (36.67\%, n=55) (Figure 3A).

Likewise, the poultry farm samples were obtained principally from turkey
barn water (66.67\%, n=20) and duck's environmental water with fecal
matter (16.67\%, n=5); and to a lesser extent from sewage, carcass wash
water, and hand-wash water (3.33\%, n=1 each one). The greatest subtype
diversity was found in environmental water of duck farms with five
subtypes: H3N2, H3N8, H4N9, H11N2, and H12N7. Only the poultry farm
along a river in China and the free-range farm in the Netherlands did
not specify the water source (Figure 3B).

The circular phylogenetic tree based on the analysis of the HA sequences
detected in water samples showed that the H1 subtype was closely related
to the H6 subtype, and the H12 to the H9 subtype (Figure 4). Likewise,
the H7 subtype had close relation with the H10 subtype, and these were
genetically close to the H3 and the H4 subtype.

The H3N2 HA sequence (Accession No: LC041327) detected from a live bird
market in Vietnam shared 95.88\% of nucleotide sequence identity with
the H3N2 HA sequence (Accession No: CY146748) from a duck farm in China.
Moreover, the H3N8 HA sequence (Accession No: CY146756) isolated from a
duck farm in the Dongting Lake region was grouped in a clade with H3N8
HA sequences from the Izumi plain in Japan. Meanwhile, H4 subtype
evidenced two geographically separated clades, the first containing
H4N6, H4N8, and H4N9 HA sequences from Asia, and the second H4N2 and
H4N6 HA sequences from America. In particular, the H4N9 HA sequence
(Accession No: CY146732) isolated from a duck farm close to the Dongting
Lake in China was related to AIVs detected in the Izumi plain, Japan
with 97.99-98.05\% nucleotide sequence identity.

All the H5N1 HA sequences were grouped together, except for one H5N1 HA
sequence (Accession Ndeg: KX247930) detected in a live bird market in
China, which was related to H5N6 HA sequences from a Kodai pond and the
Izumi plain in Japan, as well as, from a live bird market in China with
95.95-96.34\% nucleotide sequence identity. Similarly, all the H5N8 HA
sequences detected in wild bird habitats were grouped in the same clade,
except the H5N8 HA sequence (Accession No EPI553362) detected in the
Izumi plain in Japan which was phylogenetically related to the H5N2 HA
sequences detected in turkey barns from the United States with 97.95 -
98.30\% nucleotide sequence identity.

The phylogenetic analysis also showed that all the H9N2 HA sequences
were grouped together in two clusters by country (China and Bangladesh)
and were closely related to the H12N7 HA sequence (Accession No:
CY146764) detected from a duck farm located in the Dongting Lake region
in China. Finally, H11N9 HA sequences from live bird markets and the
H11N2 HA sequence from a duck farm close to a wetland in China were
clustered in the same clade with 95.58-95.70\% nucleotide sequence
identity.

\textbf{Discussion}

This paper describes the global AIV subtype diversity and distribution
in water. It was evidenced that wild bird habitats host the highest
subtype diversity reported in water samples. Wild birds of aquatic
environments such as the Anseriformes and Charadriiformes constitute the
major natural AIV reservoir. At least 105 wild bird species of 26
different families host LPAI, and almost all the AIV subtypes have been
detected in wild aquatic bird reservoirs (Olsen et al., 2006). Wild
waterfowl greatly contribute to the geographical spread of AIV subtypes
between wetlands through migratory movements associated with high bird
densities and increased contact rates among bird species (van Dijk et
al., 2018).

Globally, a total of 112 AIV subtypes have been identified in wild birds
of which 49 have been found in domestic birds (Olson et al., 2014). Only
in China, twelve subtypes of HA (H1-H12) and eight subtypes of NA
(N4-N9) of 21 different combinations have been identified in wild birds
(Tang et al., 2020). In our analysis, we evidenced that 21 subtypes were
reported from water samples, of which eleven were detected in wild bird
habitats, mainly from Asian countries. Large parts of East Asia and all
Southeast Asia comprise the East Asian-Australasian Flyway. This flyway
supports the greatest diversity and highest number of migratory birds
worldwide (Tang et al., 2020). Likewise, a large number of wild aquatic
birds with a great potential to serve as carriers of AIV migrate to
overwintering habitats in Asia annually (Deng et al., 2013; Khalil et
al., 2020; Nakagawa et al., 2018). Nevertheless, it is well-known that
geographical variations on existing surveillance efforts can also
potentially influence the AIV detection and its spatial distribution
(Berger et al., 2018).

The Izumi plain and the Dongting Lake are recognized as prominent
overwintering sites in Japan and China, respectively (Nakagawa et al.,
2018; Zhang et al., 2011). These stopover areas for several tens of
thousands of migratory birds represented the wild bird habitats with the
highest number of HA sequences and the greatest subtype diversity,
mainly the Izumi plain; wherein, six different subtypes (H3N8, H4N6,
H4N8, H5N6, H5N8, and H6N2) were reported in water samples. Moreover, in
our phylogenetic tree, most of the viruses from the Izumi plain were
related to viruses from poultry farms of the Dongting Lake region in
China and from turkey barns in the United States, as well as from
Chinese live bird markets. Previous phylogenetic analyses of AIV strains
isolated from the Izumi plain have revealed genetic reassortments
between AIV from East Asian, European, African, and North American
countries (Khalil et al., 2020, 2021; Nakagawa et al., 2018; Okuya et
al., 2015). This trans-hemispheric genetic flow of AIV highlights the
wild-domestic bird interfaces as relevant areas for influenza A virus
surveillance (Prosser et al., 2013).

The Izumi plain and the Dongting Lake are also well-known for free-range
farming and mixing between chickens and domestic aquatic fowl (Khalil et
al., 2020). In our analyses, all the HA sequences from the subtypes
H3N2, H3N8, H4N9, H11N2, and H12N7 isolated in water samples from
poultry farms in China were close to aquatic environments. Domestic
farming in Southern Asia commonly occurs with a lack of biosecurity
measures and close contact among wild waterfowls and domestic fowls that
facilitates multiple genetic reassortments (Deng et al., 2013; Zhang et
al., 2011). This type of farming is widespread in low-income countries
where the majority of poultry is raised under extensive conditions by
family-based smallholder farms (Gilbert et al., 2015). Therefore,
regions such as Eastern Europe, Central America, and sub-Saharan Africa
also pose a high potential for AIV interspecies transmission; however,
several high-risk areas have inadequate influenza A virus surveillance
(Berger et al., 2018).

Likewise, live bird markets where different domestic and wild bird
species often share the same water, food, and housing also represent an
opportunity for interspecies transmission and viral genetic
diversification (Zhang et al., 2011). In our analyses, most of the HA
sequences were detected in water samples from live bird markets, mainly
from H9N2 and H5N1 subtypes in Asia (China and Bangladesh). For more
than a decade, Asian countries have undertaken numerous efforts to
rapidly detect and track AIVs mainly for LPAI H9N2 and HPAI H5N1 through
annual surveillance programs in poultry-related environments such as
live poultry markets, poultry farms, slaughterhouses, and wild bird
habitats (Rimi et al., 2019; Zhang et al., 2019). These efforts provide
valuable information to inform decision-making and implement risk
mitigation strategies. Unfortunately, other countries and regions do not
have the same level of surveillance or do not share that data publicly,
which hampers the possibility to better understand AIV transmission
dynamics locally and globally (Chan et al., 2010).

The H5N1 virus continues to pose an important public health threat in
East and Southeast Asian countries and has become endemic in domestic
poultry in these countries (Rimi et al., 2019). Although HPAI H5N1 has
also caused many outbreaks with severe illness in poultry from Europe
and Africa (Chowdhury et al., 2019), in our analyses HPAI H5N1 viruses
were only reported in water samples from Asian countries. Similarly, in
our study LPAI H9N2 viruses were isolated solely in water samples from
Asia. Nevertheless, LPAI H9N2 avian influenza viruses have widespread in
domestic poultry worldwide (Peacock et al., 2019).

Multiple AIV subtypes have been detected from poultry and wild birds in
Africa, Australia, Europe, and America (Alexander, 2007; Araujo et al.,
2018; Brown, 2010; Grillo et al., 2015; Hansbro et al., 2010;
Jimenez-Bluhm et al., 2018; Senne, 2007). However, our global analysis
of AIV evidenced a lack of sequences isolated from environmental water
in Africa, Oceania, and South America, as well as, in Europe. Data on
influenza in tropical countries remain scarce compared with temperate
countries (Moura, 2010). Likewise, according to Moura (2010), the
emergence of AIV strains in Asia may occur and be detected approximately
6 to 9 months earlier than in Oceania, North America, and Europe, and 12
to 18 months earlier than in South America.

It is also recognized that the prevalence and distribution of avian
influenza in most tropical countries are mostly unknown as a result of
the lack of a rigorous surveillance system (Yazdanbakhsh \& Kremsner,
2009). Since the H1N1 pandemic, it has been highlighted a considerable
number of shortcomings on global epidemiological surveillance. The
absence of routine AIV monitoring has resulted in substantial
information gaps in large areas of the world, mainly from less-resourced
countries (Briand et al., 2011).

Environmental sampling has been effectively used for AIV surveillance
since the 1970s. Nevertheless, the methods and protocols are not
completely standardized, as well as, an international guideline about
data management is absent (Hood et al., 2020). This was one of the most
noticeable limitations in our analyses since several AIV no specified
the type of environment sampled (i.e., feces, water, air, mud, or
surface swabs), and thus, those HA sequences were excluded. Likewise, we
only analyzed the sequences with complete subtypes reported in the four
recognized electronic databases. In the case of Europe, there were
reported 83 sequences of H1 genes from ice and water in high-latitude
lakes visited by large numbers of migratory birds in Siberia.
Nevertheless, those sequences did not meet the inclusion criteria. We
also found several geographical regions with missing subtype data.
Therefore, the AIV subtypes described in this work may not be the exact
reflection of the global subtype diversity, but it highlights the
potential value of using this information to better understand AIV local
and global transmission dynamics. Our work also highlights the need to
improve surveillance efforts in many regions as well as to advance
towards more unified data collection and sharing standards to improve
influenza A virus surveillance and better prevent future potential
pandemics.

In conclusion, this descriptive and phylogenetic analysis of AIVs
isolated in water samples from sites at high risk for influenza
outbreaks, such as live bird markets, poultry farms, and wild bird
habitats is valuable to provide an overview and baseline information of
the current data on global AIV diversity and distribution since 2003.
However, this study highlights the need to continuing generating,
expanding, and sharing precise and detailed environmental data from
surveillance systems to allow a better understanding of the ecology and
epidemiology of AIV, especially from low- and middle-income countries.

\textbf{Acknowledgements}

This research belongs to the PAPIIT-DGAPA project Ndeg IN222119. Jessica
Mateus-Anzola 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 Mexican National Council
for Science and Technology for postgraduate fellowship (CONACyT, ID
785318).

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest.

\textbf{Data Availability Statement}

Data supporting the findings of this study are included in the
supporting file. The HA sequences of avian influenza viruses are
available in the public-access databases mentioned in this manuscript.

\textbf{Ethics Statement}

No ethical approval was required as this is an article that analyses
public-access data.

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\textbf{Figure legend}

\textbf{Figure 1. AIV subtypes detected in water samples by country.} A
total of 21 subtypes from nine countries were isolated between 2003 --
2020. The H9N2 subtype was the most reported (40.17\%, n=94), followed
by the H5N1 (23.07\%, n=54) and the H5N6 (8.97\%, n=21). The world map
shows the number of AIV subtypes reported by country; red, orange, and
yellow areas indicate high, medium, and low subtype diversity,
respectively. The map was created online at\emph{https://mapchart.net}.

\textbf{Figure 2.} \textbf{The number of} \textbf{AIV sequences detected
in water samples by sampling site.} A) live bird markets (LBM), B) wild
bird habitats (WBH), C) poultry farms (PF), D) unknown (UNK) sampling
site. Red, orange, and yellow areas in the map indicate high, medium,
and low number of AIV sequences, respectively. The pie charts show the
proportion of each subtype by country in each sampling site. The bar
chart presents the number of AIV sequences by country and sampling site.
The map was created online at\emph{https://mapchart.net}.

\textbf{Figure 3. Number of AIV sequences by water source and
subtype.}A) Live bird market, B) Poultry farm. Carcass-wash water (CWW),
duck's environmental water (DEW), drinking water trough (DW), hand-wash
water (HWW), poultry cage water (PCW), sewage (SW), turkey barn water
(TBW), and unknown (UNK). The highest subtype diversity was found in DW
from live bird markets, and in DEW from a poultry farm.

\textbf{Figure 4.} \textbf{Circular phylogenetic tree of hemagglutinin
(HA) gene sequences of AIV detected in water samples between 2003 --
2020 worldwide} . Viral sequences are color-coded by subtype. Branches
are color-coded by sampling site (red= live bird market (LBM), blue=WBH
(wild bird habitat), purple= poultry farm (PF), and black= unknown
(UNK)). The tree was generated with the Maximum-likelihood method using
MEGA X and edited with FigTree v1.4.4 software.

\textbf{Supporting information}

\textbf{Table S1.} Accession numbers of AIV hemagglutinin (HA) gene
sequences detected in water samples from 2003 -- 2020.\selectlanguage{english}
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