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# Influenza A (H1N1): Cross-Species Variant Analysis ## Evidence 2 – BT1013 Emerging Viruses | April 2026 **Authors:** Charlize Arriaza & Santiago Martínez **Student ID:** A00844457 & A01287509 **Institution:** ITESM --- ## Overview This analysis examines **20 complete H1N1 hemagglutinin (HA) gene sequences** across four host species—humans, swine, avian, and canine—to investigate cross-species transmission patterns, genetic diversity, and pandemic risk. The study directly addresses **Case 2: Variants Between Species**, exploring which animal populations serve as viral reservoirs and how frequently the virus jumps between host species. --- ## Research Questions 1. **Do viruses from different host species form separate evolutionary groups, or are they genetically interleaved?** - *What does this tell us about cross-species transmission frequency?* 2. **Which animal species shows the greatest H1N1 variant diversity?** - *Is there evidence of sustained, independent transmission in that population?* 3. **If animal populations develop unique mutations absent in humans, what are the implications for long-term epidemic control?** - *How should we address natural viral reservoirs?* --- ## Methods ### Data Collection - **20 H1N1 HA sequences** retrieved from GenBank (NCBI) via `rentrez` package - **Strains:** 6 human, 6 swine, 4 avian, 4 canine isolates - **Accessions:** Verified complete CDS from NIAID Influenza Sequence Database (CY-prefix) and GenBank - **All sequences:** Exactly 1,701 nucleotides (start codon to stop codon) ### Computational Analysis - **Sequence alignment:** DECIPHER (progressive pairwise alignment) - **Genetic distance:** Jukes-Cantor (JC69) model via `ape::dist.ml()` - **Phylogenetic reconstruction:** Neighbor-joining tree with midpoint rooting - **Sequence variability:** Shannon entropy analysis to identify variable regions - **Visualization:** `ggplot2`, `ggmsa`, `reshape2` for publication-quality figures ### Software & Packages - **R version:** 4.x - **Key packages:** - `rentrez` — NCBI database access - `Biostrings` — DNA sequence manipulation - `DECIPHER` — Multiple sequence alignment - `ape` — Phylogenetics - `phangorn` — Tree building - `ggplot2` — Data visualization --- ## Key Findings ### 1. **Cross-Species Transmission is Frequent** Human and swine H1N1 sequences are **genetically interleaved** on the phylogenetic tree (Figure 8), rather than forming exclusive, host-specific clusters. This pattern indicates **bidirectional transmission** between humans and pigs occurs regularly and at high enough frequency to leave a genetic signature. Canine sequences cluster with 2009 human pandemic strains, suggesting recent human-to-dog spillover. Avian sequences form a more distinct outgroup, reflecting a higher (but not impenetrable) species barrier. ### 2. **Swine Are the Most Vulnerable Host** Swine populations harbor the greatest H1N1 variant diversity globally, with **three major co-circulating lineages:** - Classical swine lineage (direct descendant of 1918 pandemic virus) - Triple-reassortant lineage (emerged 1990s in North America) - Eurasian avian-like lineage (circulates in Europe and Asia) **Evidence of sustained transmission:** Rajão et al. (2016, *PLOS Pathogens*) documented **continuous H1N1 diversification in U.S. swine herds over 15 years**, with multiple independent human-to-swine spillover events generating new lineages that then circulated independently for years. This is the hallmark of a true animal reservoir. ### 3. **Animal Reservoirs Pose Long-Term Pandemic Risk** Unique mutations in swine H1N1 populations (not present in humans) indicate **independent evolutionary pressures** without human immune selection. If such a variant spills back into humans, existing immunity (from prior infection or vaccination) may offer **reduced protection**, creating pandemic potential. This was precisely the mechanism behind the 2009 pandemic. --- ## Proposed Solutions: One Health Framework To control H1N1 at the animal-human interface, we propose a **three-pillar One Health approach:** ### 1. **Integrated Genomic Surveillance** - Real-time sequencing of H1N1 in swine and poultry populations - Data feed directly into WHO influenza monitoring networks - Not limited to human clinical cases ### 2. **Targeted Vaccination in Animal Populations** - Vaccinate high-risk pig populations in regions of close human-animal contact - Priority regions: Southeast Asia, sub-Saharan Africa, rural Latin America ### 3. **Enhanced Farm Biosecurity** - Reduce conditions allowing multiple strains to co-infect the same animal - Prevent reassortment (exchange of gene segments between strains) - Improve housing, hygiene, and isolation protocols --- ## Data & Reproducibility ### Strain Metadata (20 H1N1 Isolates) | Host | Strain | GenBank Accession | HA Length (nt) | |------|--------|-------------------|---| | **Human (6)** | | | | | | A/California/04/2009 | FJ966082 | 1,701 | | | A/California/07/2009 | FJ966974 | 1,701 | | | A/Puerto_Rico/8/1934 | CY034139 | 1,701 | | | A/Brisbane/59/2007 | CY031391 | 1,701 | | | A/South_Carolina/1/1918 | AF117241 | 1,701 | | | A/New_Caledonia/20/1999 | AY289929 | 1,701 | | **Swine (6)** | | | | | | A/swine/Iowa/15/1930 | CY099151 | 1,701 | | | A/swine/Hong_Kong/9745/2001 | AY619961 | 1,701 | | | A/swine/Nebraska/1/1992 | CY098528 | 1,701 | | | A/swine/North_Carolina/18161/2002 | EU604689 | 1,701 | | | A/swine/Minnesota/593/2009 | GU734048 | 1,701 | | | A/swine/Iowa/1/1985 | CY098711 | 1,701 | | **Avian (4)** | | | | | | A/duck/Alberta/35/1976 | CY096744 | 1,701 | | | A/mallard/Alberta/1/1976 | CY096736 | 1,701 | | | A/duck/New_York/1996 | AF084263 | 1,701 | | | A/duck/Bavaria/1/1977 | AF084260 | 1,701 | | **Canine (4)** | | | | | | A/canine/Colorado/1/2013 | CY147366 | 1,701 | | | A/canine/Illinois/12191/2015 | CY163351 | 1,701 | | | A/canine/Zhejiang/01/2010 | JF789617 | 1,701 | | | A/canine/Beijing/2009 | HM046011 | 1,701 | **All sequences are complete coding sequences (CDS) from start codon to stop codon.** --- ## Figures **Figure 1:** HA gene sequence lengths across 20 H1N1 strains by host species **Figure 2:** GC content (%) — sequence composition patterns by host **Figure 3:** Nucleotide composition (A, C, G, T) stacked by strain **Figure 4:** Shannon entropy profile — identifies variable genomic regions **Figure 5:** MSA Region 1 (receptor-binding domain) — alignment positions 1–110 **Figure 6:** MSA Region 2 (HA stalk/HA2 region) — alignment positions 2,686–2,785 **Figure 7:** Pairwise genetic distance heatmap (JC69 model) — strains ordered by host **Figure 8:** Neighbor-joining phylogenetic tree (midpoint-rooted) — host species colored --- ## References Centers for Disease Control and Prevention. (2024). *Influenza A (H1N1) overview.* https://www.cdc.gov/flu Dawood, F. S., et al. (2012). Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation. *The Lancet Infectious Diseases*, 12(9), 687–695. https://doi.org/10.1016/S1473-3099(12)70121-4 Garten, R. J., et al. (2009). Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. *Science*, 325(5937), 197–201. https://doi.org/10.1126/science.1176225 Paradis, E., & Schliep, K. (2019). ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R. *Bioinformatics*, 35(3), 526–528. https://doi.org/10.1093/bioinformatics/bty633 Rajão, D. S., et al. (2016). Novel reassortant human-like H3N2 and H3N1 influenza A viruses detected in pigs are virulent and antigenically distinct from swine viruses endemic to the United States. *Journal of Virology*, 89(22), 11213–11222. https://doi.org/10.1128/JVI.01675-15 Schliep, K. P. (2011). phangorn: Phylogenetic analysis in R. *Bioinformatics*, 27(4), 592–593. https://doi.org/10.1093/bioinformatics/btq706 Smith, G. J. D., et al. (2009). Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. *Nature*, 459, 1122–1125. https://doi.org/10.1038/nature08182 Taubenberger, J. K., & Morens, D. M. (2010). Influenza: The once and future pandemic. *Public Health Reports*, 125(Suppl 3), 16–26. Wickham, H. (2016). *ggplot2: Elegant graphics for data analysis* (2nd ed.). Springer-Verlag. https://doi.org/10.1007/978-3-319-24277-4 Winter, D. J. (2017). rentrez: An R package for the NCBI eUtils API. *The R Journal*, 9(2), 520–526. https://journal.r-project.org/archive/2017/RJ-2017-058/ World Health Organization. (2023). *Global influenza strategy 2019–2030.* WHO Press. https://www.who.int/publications/i/item/9789240011878 --- ## Course Context **Course:** BT1013 — Emerging Viruses **Assignment:** Evidence 2 — Cross-Species Variant Analysis (Case 2) **Learning Outcomes:** - Analyze viral sequence data using R bioinformatics tools - Construct and interpret phylogenetic trees - Identify patterns of cross-species transmission - Propose evidence-based public health interventions --- ## Code Availability The complete R Markdown source code for this analysis, including all data processing, visualization, and statistical steps, is available in the accompanying `.Rmd` file. Code is fully commented and reproducible. **All analyses can be reproduced by running the R code chunks in order.** --- ## How to Cite This Work **APA Format:** ``` Arriaza, C., & Martínez, S. (2026, April). Influenza A (H1N1): Cross-species variant analysis. *BT1013 Emerging Viruses*. RPubs. https://rpubs.com/[Santiago_Mtz_Cpd]/[DocumentID] ``` **In-text citation:** According to Arriaza and Martínez (2026), H1N1 exhibits significant cross-species transmission patterns, with human and swine sequences genetically interleaved on phylogenetic trees. --- ## Contact For questions about this analysis, please contact: - **Charlize Arriaza:** A00844457@tec.mx - **Santiago Martínez:** A01287509@tec.mx --- **Last updated:** April 2026 **Analysis completed:** April 2026 **Published:** RPubs, May 1st 2026