nouvelles de l'entreprise MDPI | Novel H1N1 Swine Influenza Virus Discovered in Shandong Pig Herd: Analysis of Cross-Species Transmission Risk and

On January 15, 2026, Professor Yu Zhijun's team from the College of Biological Science and Technology, Jinan University, in collaboration with the Poultry Research Institute of the Shandong Academy of Agricultural Sciences and Shandong Normal University, published a research paper entitled "Genetic Characterization and Evolutionary Insights of Novel H1N1 Swine Influenza Viruses Identified from Pigs in Shandong Province, China" in the international virology journal *Viruses* (MDPI).

This study detected two H1N1 swine influenza viruses (SD6591 and SD6592) in pig samples from Linyi City and Shanghe County, Jinan City, Shandong Province in 2019, and completed their whole-genome sequencing and phylogenetic analysis. The study found that the PA and M2 genes of the SD6591 strain showed high homology with human H1N1 virus, suggesting that this strain may exhibit human-swine influenza virus gene reassortment characteristics. The SD6592 strain, however, was highly homologous to swine influenza virus, showing no obvious cross-host gene reassortment characteristics, and its genetic characteristics were relatively conserved.

This study provides molecular epidemiological evidence for understanding the cross-species evolution of swine influenza viruses in Shandong Province, emphasizing the importance of continuously strengthening swine influenza surveillance and zoonotic risk assessment.

Research Highlights

* **Reveal of a Novel Human-Pig Reassortant Genotype:** Genomic analysis of the SD6591 strain showed that its PA and M2 genes are highly homologous to human seasonal H1N1 viruses, while other genes are mainly derived from swine viruses, indicating that it is a novel human-swine reassortant virus.

* **Implication of a Complex Cross-Species Transmission Network:** Phylogenetic analysis revealed that the M2 gene of SD6591 clusters in the same evolutionary branch as canine H1N1 viruses, suggesting a possible multi-host cycle between pigs, dogs, and humans, highlighting the necessity of including companion animals in the surveillance system.

* **Confirmation of Potential Human Cell Infection Capability:** In vitro experiments with highly homologous active strains confirmed that the virus can replicate efficiently in human lung epithelial cells (A549), providing direct experimental evidence for its potential zoonotic risk.

• An innovative functional validation strategy was adopted: In response to the challenge of failing to isolate live viruses due to sample preservation, the study innovatively used existing strains with high genomic homology (>91%) for in vitro functional substitution validation, providing a feasible solution for virus characteristic studies under similar circumstances.

This study conducted nucleic acid testing for H1N1 subtype swine influenza virus on 200 pig lung tissue samples collected in 2019 from Liaocheng, Linyi, Jinan, and Qingdao in Shandong Province. Two positive samples (SD6591 and SD6592) were detected. The complete viral genome sequence was obtained through high-throughput sequencing, and its genetic evolutionary characteristics were systematically analyzed using homology comparison and phylogenetic analysis. This study aims to provide basic data for molecular epidemiological surveillance of swine influenza virus in Shandong Province and to provide a scientific basis for assessing its cross-species transmission risk and formulating targeted prevention and control strategies.

Introduction

Influenza A viruses have a wide host range, infecting multiple species including humans, birds, and pigs. Swine influenza virus (SIV) not only causes significant economic losses to the pig farming industry but also poses a persistent threat to public health due to the unique biological role of pigs as "mixing vessels" for influenza viruses, which allows them to continuously generate novel recombinant viruses and spread across species.

Research Results

1. SD6591 exhibits unique human-derived gene reassortment characteristics:

The PA gene of strain SD6591 shows the highest homology (96.99%) with the human 2009 H1N1 virus. While the M2 gene shows the highest homology (98.96%) with the swine-derived Liaoning H1N1 strain, its gene cluster is highly correlated with human seasonal H1N1 viruses. Six gene segments, PB2, PB1, NP, NA, HA, and NEP, are highly homologous to classic swine H1N1 viruses. This "6+2" gene combination pattern suggests that the virus may be a product of gene reassortment between human and swine influenza viruses. Notably, the M2 gene shows affinity for both swine and canine H1N1 viruses (A/canine/Korea/MV1/2012) on the phylogenetic tree and clusters in the same evolutionary branch, suggesting that this gene may have entered the swine population through canine-swine cross-species transmission.

Table 1. Strains with the closest homology to SD6591 for each gene.

2. SD6592 maintains the genomic characteristics of classic porcine H1N1 virus:

Unlike SD6591, all eight gene segments of the SD6592 strain share over 98% homology with porcine H1N1 virus, and are most closely related to the 2018 Beijing isolate A/swine/Beijing/0301/2018 (H1N1). This highly conserved genomic characteristic indicates that this strain has established a stable transmission chain in the local pig population and occupies a persistent ecological niche.

Table 2. Strains with the highest homology to SD6592.

3. No significant recombination signal detected in recombination analysis:

SimPlot analysis suggested chimeric-like features in the PA gene of SD6591 and the NA and NP genes of SD6592. However, further verification using multiple algorithms in the RDP5 software package, including RDP, GENECONV, and BootScan (with a corrected p-value threshold <0.05), showed no statistically significant recombination signal. The related sequence differences are more likely due to the gradual accumulation of point mutations or unsampled ancestral sequences, indicating that there is currently no clear evidence to support the existence of a recent recombination event in this lineage.

Figure 1. Results of SD6591 PA gene recombination analysis.

Figure 2. Results of recombination analysis of SD6592 NA and NP genes.

4. Highly Homologous Substitutes Possess Multi-Host Cell Replication Capability:

The research team conducted in vitro functional validation using a 2018-H1N1 isolate (A/swine/China/Qingdao/2018) with >91% genomic homology to SD6591/SD6592. Western blot and qRT-PCR results showed that the virus could replicate efficiently in porcine (PK-15), canine (MDCK), and human (A549) epithelial cells, with replication kinetics similar to the control strain PR8. These results suggest at the cellular level that this type of swine influenza virus has the potential to infect cells from multiple host sources.

Table 3. Nucleotide identity (%) between the SD6591 gene fragment and two reference H1N1 strains.

Table 4. Nucleotide identity (%) between the SD6592 gene fragment and two reference H1N1 strains.

Figure 3. Replication kinetics of 2018-H1N1 and PR8 viruses in different cell lines.

5. Evolutionary characteristics of the M2 gene suggest attention to drug resistance-related sites:

The M2 gene of SD6591 shows the highest homology (97.93%) with the 2009 pandemic H1N1 virus and clusters in the same evolutionary branch as canine H1N1 virus (A/canine/Korea/MV1/2012) in phylogenetic analysis. Previous studies have shown that human influenza viruses in this evolutionary branch commonly carry key mutation sites associated with amantadine resistance (such as S31N). This genetic background suggests the need to pay attention to the potential drug resistance-related molecular characteristics of the SD6591 strain, and further experimental verification of key sites and functions of the M2 protein is necessary.

Summary

This study completed the whole-genome analysis of two H1N1 swine influenza viruses from Shandong Province. Utilizing an innovative "homologous virus functional substitution" strategy, we correlated their sequence characteristics with their in vitro replication phenotype, confirming that the H1N1 swine influenza virus circulating in pig populations still possesses the potential for cross-species multi-host cell infection even in the absence of detectable significant gene recombination events. This finding underscores the crucial importance of strengthening swine influenza virus genome surveillance and validating the phenotypic characteristics of representative strains for timely detection of potential adaptive mutations and early warning of novel influenza outbreaks.