In 2009 a new influenza virus jumped from pigs to humans and spread very rapidly, causing an initial outbreak in Mexico and becoming a global pandemic in just a few months. Although the most straightforward explanation is that the virus originated in swine in Mexico, several studies suggested that this was unlikely because key genetic components of the virus had never been detected in the Americas. Determining the source of the disease is critical for predicting and preparing for future influenza pandemics.

Mena, Nelson et al. sought to better characterize the genetic diversity of influenza viruses in Mexican swine by obtaining the entire genetic sequences of 58 viruses collected from swine in Mexico, including some from previously unsampled regions in central Mexico. The sequences revealed extensive diversity among the influenza viruses circulating in Mexican swine. Several viruses included genetic segments that originated from viruses from Eurasia (the landmass containing Europe and Asia) and had not previously been detected in the Americas. The new sequences contained key genetic components of the 2009 pandemic virus. Furthermore, the sequences suggest that viruses with a similar genetic composition to the 2009 pandemic virus have been circulating in pigs in central-west Mexico for more than a decade. Thus, this region is the most likely source of the virus that started the 2009 pandemic.

Mena, Nelson et al. also found that the movement of viruses from Eurasia and the United States into Mexico closely follows the direction of the global trade of live swine. This highlights the critical role that animal trading plays in bringing together diverse viruses from different continents, which can then combine and generate new pandemic viruses.

A potential next step is to perform experiments that investigate how well the swine viruses can replicate and pass between different animal models. Comparing the results of such experiments with the findings presented by Mena, Nelson et al. could identify factors that make the viruses more likely to spread to humans and produce a pandemic.

Our ability to predict outbreaks of zoonotic pathogens requires an understanding of their ecology and evolution in reservoir hosts. At the onset of the 2009 influenza pandemic, whole-genome sequencing revealed that pdmH1N1 was a novel reassortant virus comprised of segments from three major swIAV lineages (Garten et al., 2009): segments PB2, PB1, PA, NP and NS were derived from a triple reassortant H3N2 swine virus (TRsw) that originated in North American swine during the mid-1990s; the HA (H1) segment derived from the classical swine H1N1 lineage (Csw) that has circulated in North American swine since the 1918 pandemic; and the NA (N1) and MP segments were related to the avian-like Eurasian swine lineage (EAsw) that emerged in European pigs in the late 1970s. Genetic and epidemiological evidence indicate that the first outbreak of pdmH1N1 occurred in humans in Mexico (Brockmann and Helbing, 2013; Chowell et al., 2011; Lemey et al., 2009). However, it remained unclear whether Mexico also was the site of the zoonotic transmission event that gave rise to the 2009 outbreak, given that EAsw viruses had never been detected in swine in any part of the Americas, including Argentina, Brazil, Chile, Mexico, Peru, Canada, and the intensively sampled swine herds in the United States (Anderson et al., 2013; Nelson et al., 2015b; Pereda et al., 2011; Tinoco et al., 2015). At the time, Asia was the only region where TRsw, Csw, and EAsw viruses were known to co-circulate in swine (Zhu et al., 2013).

Asia is a global source of novel human seasonal influenza viruses (Lemey et al., 2014; Russell et al., 2008), avian viruses associated with the pandemics of 1957 and 1968 (Kawaoka et al., 1989), and emerging avian viruses with pandemic potential (Lam et al., 2015; Taubenberger Morens, 2009). Since 2009, several studies have implicated Asian swine as a possible source of the TRsw/Csw/EAsw reassortant swine virus that gave rise to pdmH1N1. Asia is the only region where TRsw, Csw, and EAsw are known to exchange segments through reassortment (Lam et al., 2011; Poonsuk et al., 2013; Takemae et al., 2008; Vijaykrishna et al., 2011). At the onset of the pandemic, IAVs identified in swine (swIAVs) in Hong Kong, SAR, were more closely related to pdmH1N1 than any other swIAVs available globally (Smith et al., 2009). One Hong Kong swIAV was identified with a genotype similar to the pdmH1N1 virus except for the NA segment, and transmitted via respiratory droplet in ferrets (Yen et al., 2011). However, the time of the most recent common ancestor (tMRCA) of the Hong Kong swIAVs and pdmH1N1 was still approximately 10 years (Smith et al., 2009). Combined with the lack of any complete swIAV genomes from Mexico’s large swine populations at the start of the pandemic, and the difficulty of understanding how a virus that evolved in Asian swine caused its first outbreak in humans in Mexico, the geographical location of the swine-to-human transmission event that gave rise to pdmH1N1 has remained uncertain. In the years following the pandemic, new surveillance in Mexican swine has identified Csw, TRsw, and pdmH1N1 and seasonal H3N2 viruses of human origin (Nelson et al., 2015a), but no EAsw viruses. However, the majority of swIAVs in previous analyses were collected in northern and eastern Mexico, and no studies to date have included viruses from major swine-producing states in central Mexico, including Jalisco, Guanajuato, and Puebla (Figure 1).

Figure 1

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Figure 1—source data 1

Swine population sizes in Mexican states in 2014.

Data available from Mexico's Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA) (www.siap.gob.mx).

https://doi.org/10.7554/eLife.16777.004

Download elife-16777-fig1-data1-v2.csv

Resolving whether the viral precursors of pdmH1N1 evolved in swine in Mexico or Asia has important implications for understanding pandemic emergence and informing risk assessment. We therefore undertook expansive surveillance efforts in Mexico, isolating the virus from pigs with respiratory symptoms in farms from six Mexican states with high swine production, including Sonora in northern Mexico, Yucatan in eastern Mexico, and previously unsampled states in central-east Mexico (Puebla), and central-west Mexico (Jalisco, Guanajuato, and Aguascalientes, Figure 1). These efforts yielded 58 complete viral genomes (sequence accession numbers are available in the Dryad data repository under Doi:10.5061/dryad.m550m) that were used to identify swIAVs that are closely related to pdmH1N1, resolving the origins of pdmH1N1 in its reservoir host.

Overall, our findings resolve a long-standing question of where the pandemic H1N1 virus originated in swine. Moreover, they underscore the importance of understanding the global evolution of IAVs in pigs, and the pandemic threat presented by the large number of independently evolving swIAV populations worldwide that periodically experience invasions of new viruses. Our findings demonstrate the central importance of inter-hemispheric swine movements in the long-range dissemination of swIAV diversity, which allowed divergent Eurasian and North American viruses to co-circulate in central Mexico and reassort into genotype 1 viruses (Figure 8). It remains unclear whether conditions in Mexico were uniquely suitable for the emergence of genotype 1 viruses, or whether it is possible that similar viruses also circulate undetected in Asia, the only other location where Csw, EAsw, and TRsw viruses and various reassortants are known to be present (Lam et al., 2011). The inability of researchers to produce genotype 1 viruses experimentally in cell cultures or pigs (Ma et al., 2014) implies strain-specific differences in reassortment capabilities. The likelihood of pandemic emergence is therefore enhanced by the seeding of diverse swIAV lineages in countries with varying swine production practices and fitness pressures. The independent evolution of multiple variants increases the probability that one will evolve the capacity to transmit to humans.

Figure 8

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Although understanding the positive and negative interactions between fitness in human and swine hosts is central to understanding pandemic emergence, this area of research remains poorly understood. Strong patterns of reassortment between EAsw and North American viruses in Mexican swine are an indication of competitive interactions. The apparent fitness of the TRIG + EAsw MP internal gene constellation in central Mexican swine was central to the emergence of the genotype 1 viruses that gave rise to pdmH1N1, but is not well understood. The success of genotype 1 viruses for many years in central-west Mexico is notable, given the low levels of persistence of pdmH1N1 viruses as an intact genome in swine following reverse zoonosis in many countries (Liang et al., 2014; Nelson and Vincent, 2015; Nelson et al., 2015d; Pereda et al., 2011; Watson et al., 2015), a pattern also observed in Sonora. The lower levels of onward transmission of pdmH1N1 surface genes in swine could relate to competition with existing H1s and N1s. The identification of EAsw NA and MP segments in central Mexican swine is notable, given that these segments were associated with enhanced respiratory droplet transmission in animal models (Lakdawala et al., 2011; Yen et al., 2011). The EAsw MP segment also is associated with zoonotic transmission of swIAVs at agricultural fairs in the US (Bowman et al., 2012). A number of studies have examined the transmission potential of naturally occurring or lab-generated avian and swine viruses in ferret or other animal models, as a measure of pandemic potential (Herfst et al., 2012; Sorrell et al., 2009; Yang et al., 2016). However, a fundamental understanding of fitness landscapes and trade-offs in multi-host systems has been constrained by the a lack of final resolution on the ethics of various experiments that involve novel viruses that present a potential threat to humans (Frank et al., 2016). The identification of viruses related to the 2009 pandemic precursors invites new experimental research into central questions of pandemic emergence at the human-swine interface.

The detection of EAsw in Mexico suggests that even relatively low levels of livestock movement can disseminate a highly transmissible virus such as IAV long distances, bringing attention to the importance of surveillance and mitigation strategies, including quarantine. Presently, imported live swine are not routinely tested for influenza. The emergence of the 2009 pandemic virus was closely linked to the increase in Mexico's imports of live swine during the 1990s and the influx of new influenza virus lineages from the United States and Europe. At this time we cannot rule out Asia-to-Mexico viral migration, owing to possible omissions in reported imports and exports of swine and gaps in sequencing of viruses in swine globally. However, all that is known about the nature of international swine production and trade suggests that this direction of viral movement is unlikely. Long-distance trade of pigs from Europe, the United States, and Canada to countries in Asia and South America occurs primarily to import high-efficiency European and North American breeding lines. Although illegal trade of poultry is important in the spread of AIVs, particularly within the East Asian region, there are no known economic incentives (and strong economic disincentives) for the illegal trade of pigs from Asia and Mexico. Globally, Asia is overwhelmingly a net importer of swine from Europe and North America, with extremely little export of Asian pigs or their viruses to any other continents (Nelson et al., 2015c). Another outstanding question is whether genotype 1 viruses have disseminated from Mexico to other countries in Latin America, where surveillance is low or non-existent. Mexico has reported export of live swine to several countries in Latin America (Belize, Colombia, Costa Rica, Cuba, El Salvador). However, the total volume of Mexico’s live swine exports during 1996–2012 amounts to only one-tenth of Costa Rica’s over the same time period, and one-thirtieth of Brazil’s. Mexico is therefore considered an ecological sink for swine influenza viruses, with limited opportunity for viral export to other countries (Nelson et al., 2015c).

The unexpected detection of EAsw in Mexico highlights the need to revisit outdated assumptions about the spatial distribution of IAVs in swine populations globally, given the capacity of long-distance live swine movements to disseminate viral diversity between continents. Although surveillance of swIAVs has been initiated in many new regions since 2009, it remains highly imbalanced on a global scale and low compared to avian surveillance (Nelson and Vincent, 2015). Recent detections of novel swIAVs in regions that traditionally have not conducted surveillance in swine, including Latin America and South-east Asia, underscore the importance of expanding surveillance in the large number of under-sampled regions with high densities of swine (Cappuccio et al., 2011; Nelson et al., 2015a; Ngo et al., 2012; Perera et al., 2013). China remains an important source of zoonotic influenza viruses with pandemic potential, and the identification of strong regional variations in swIAV diversity in Mexico highlights the importance of capturing swIAV diversity across China’s heterogeneous regions. Although the importance of regional live swine movements in the evolution of swIAVs in the United States has been documented (Nelson et al., 2011), many countries lack data on the intra-country movements of swine, limiting study of interactions between animal movements and regional patterns of swIAV diversity. Data on poultry movements within and between countries are similarly lacking, limiting our understanding of the role of trade in the spatial dynamics of IAVs in domestic birds. Our ability to predict future pandemics will require intensified viral surveillance and an understanding of how economic forces and international trade policies affect changes in animal movements and production practices that drive viral emergence.

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Thank you for submitting your article "Origins of the 2009 H1N1 influenza pandemic in swine in Mexico" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by Arup Chakraborty as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript presents a phylogenetic analysis of 58 full influenza A virus genomes collected from swine in Mexico to shed light on the origin of the 2009 pandemic of H1N1 influenza. The newly sequenced genomes close important gaps in our understanding of the emergence of this pandemic. The authors show that virus variants with sequences closely related to all segments of human H1N1pdm09 circulate in Mexican swine. This finding singles out Mexico as the likely region where the pandemic virus emerged. These results emphasize the need for concerted global surveillance of influenza virus diversity in animals.

All reviewers agree that the data shed light on an important and interesting problem. However, before we can recommend acceptance, a number of issues should be addressed.

Essential revisions:

1) More details on sample collection: How many samples were tested in different years? How were animals chosen for testing? Did these animals come from large commercial facilities or backyard farms? What were the criteria for sequencing positive samples? Within each location (Sonora, Puebla, etc) did the samples consistently come from the same producers/farms?

2) We would like to see a more extensive discussion of the gaps in surveillance in swine (not only in Mexico, but also Asia and Europe), and how these gaps limit the inferences that can be made.

Essential revisions:

1) More details on sample collection: How many samples were tested in different years? How were animals chosen for testing? Did these animals come from large commercial facilities or backyard farms? What were the criteria for sequencing positive samples? Within each location (Sonora, Puebla, etc) did the samples consistently come from the same producers/farms?

Following the reviewers’ suggestions, we have extended the description of the sample collection and virus isolation in the Materials and Materials and methods section to include all the information requested by the reviewers. The revised paragraph is:

“Collection of influenza viruses in Mexican swine. Swine influenza isolates were obtained from samples submitted to Avimex laboratories (Mexico) for routine confirmation diagnostics of pigs with respiratory syndrome. […] GenBank accession numbers of the 58 genomes are available in the Dryad data repository under DOI: 10.5061/dryad.m550m. Figure 2—figure supplement 1 describes the date, location, subtype and lineage assignment of each viral segment.”

2) We would like to see a more extensive discussion of the gaps in surveillance in swine (not only in Mexico, but also Asia and Europe), and how these gaps limit the inferences that can be made.

This is a very important issue, and our revised manuscript addressed this matter in further detail in the Results and the Discussion. We have also changed many of our references from ‘European swine’ to ‘Eurasian swine’ to better reflect ambiguities about the source Eurasian viruses in Mexico.

Results; “We also recognize the need to draw phylogeographic inferences with an awareness of missing data. Major gaps in surveillance in swine in Europe and Asia during the 1990s have resulted in long branch lengths between swIAVs collected in Mexico and Eurasia (Figure 3—figure supplement 1). Although it is difficult to infer from the tree whether the EAsw MP identified in Mexico was introduced from Asian or European swine (Figure 4—figure supplement 1), the lack of live swine imports reported between Mexico and any Asian country (Figure 6), and the low export of Asian swine globally (Nelson et al., 2015c) suggest that Europe may be a more likely source of the Mexican viruses, similar to the H1 and N1 segments.”

Discussion; “At this time we cannot rule out Asia-to-Mexico viral migration, owing to possible omissions in reported imports and exports of swine and gaps in sequencing of viruses in swine globally. […] Globally, Asia is overwhelmingly a net importer of swine from Europe and North America, with extremely little export of Asian pigs or their viruses to any other continents (Nelson et al., 2015c).”

Author details

1. Ignacio Mena

   1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States
   2. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   IM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Martha I Nelson

   Competing interests

   The authors declare that no competing interests exist.

2. Martha I Nelson

   Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, United States

   Contribution

   MIN, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Ignacio Mena

   Competing interests

   The authors declare that no competing interests exist.

3. Francisco Quezada-Monroy

   Laboratorio Avi-Mex, Mexico City, Mexico

   Contribution

   FQ-M, Collected and processed samples, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Jayeeta Dutta

   Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   JD, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Refugio Cortes-Fernández

   Laboratorio Avi-Mex, Mexico City, Mexico

   Contribution

   RC-F, Collected and processed samples, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. J Horacio Lara-Puente

   Laboratorio Avi-Mex, Mexico City, Mexico

   Contribution

   JHL-P, Collected and processed samples, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Felipa Castro-Peralta

   Laboratorio Avi-Mex, Mexico City, Mexico

   Contribution

   FC-P, Collected and processed samples, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Luis F Cunha

   Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   LFC, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

9. Nídia S Trovão

   1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States
   2. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, United States
   3. Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, United States
   4. Department of Microbiology and Immunology, Rega Institute, University of Leuven, Leuven, Belgium

   Contribution

   NST, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2106-1166

10. Bernardo Lozano-Dubernard

    Laboratorio Avi-Mex, Mexico City, Mexico

    Contribution

    BL-D, Collected and processed samples, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

11. Andrew Rambaut

    1. Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, United States
    2. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom
    3. Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom

    Contribution

    AR, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

12. Harm van Bakel

    Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, United States

    Contribution

    HvB, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

13. Adolfo García-Sastre

    1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States
    2. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, United States
    3. Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, United States

    Contribution

    AG-S, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    adolfo.garcia-sastre@mssm.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-6551-1827

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