Genetic Characterization and Evolutionary Insights of Novel H1N1 Swine Influenza Viruses Identified from Pigs in Shandong Province, China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents a significant contribution to influenza virology through the molecular characterization of two novel H1N1 swine influenza A virus strains (SD6591 and SD6592) identified in Shandong Province, China. The research demonstrates methodological rigor with systematic analysis of 200 porcine lung samples collected in 2019, utilizing next-generation sequencing and comprehensive bioinformatics approaches including phylogenetic analysis with MEGA11 and recombination detection using SimPlot and RDP5 software. Particularly noteworthy is the discovery that SD6591 exhibits a unique triple-reassortant genotype with PB2, PB1, NP, NA, HA, and NEP genes clustering with swine-origin H1N1 viruses, while its PA and M2 genes show human-origin characteristics, with the M2 gene additionally linked to canine influenza viruses, revealing complex cross-species transmission patterns. Meanwhile, SD6592 shows remarkable genomic conservation (>98% nucleotide homology) with previously characterized H1N1 swine variants. Although initial analyses suggested potential recombination events in PA, NA, and NP genes, rigorous validation through RDP5's multi-algorithm approach confirmed no statistically significant recombination, indicating genetic stability maintained primarily through point mutations.

I recommend acceptance of this manuscript‌ based on its novel findings and important implications for enhancing SIV surveillance and developing evidence-based prevention strategies against emerging influenza threats. The research provides critical insights into viral evolution at the human-swine interface and represents exactly the type of surveillance-based research needed for early detection of potential zoonotic threats.

The Discussion could briefly integrate existing literature to comment on the potential functional relevance of the PA and M2 gene segments identified in SD6591, such as their possible roles in viral replication efficiency, host adaptation, or antiviral drug sensitivity. This would enhance the biological interpretation of the genomic findings.

Author Response

Thank you for your careful review and valuable feedback on this article. The suggestion you pointed out about exploring the functional correlation between PA and M2 genes in depth is very relevant, which is crucial for enhancing the biological insight of this study. We have made significant additions and revisions to the discussion section of the paper based on your suggestions.

The specific modifications are as follows:

In the discussion section, we have added a paragraph (lines 309-332) specifically discussing the potential functional significance of the unique gene fragments (PA and M2) in SD6591.

Regarding the PA gene: We cited relevant literature (new reference [15]) and pointed out that the introduction of human PA gene may play a role in virus replication efficiency and mammalian host adaptation by affecting the function of polymerase complex. We discussed the implications of this discovery for the sustained presence and potential cross species transmission of the virus in pig herds, based on the results of our study.

Secondly, regarding the M2 gene: we cited relevant literature (new reference [16]), and found that the M2 ion channel protein plays a central role in the virus shedding process and is also a target of the classic antiviral drug amantadine. The phylogenetic analysis of this study reveals for the first time that the M2 gene of SD6591 strain is clustered in the same evolutionary branch as the human and canine influenza virus lineages that have been prevalent in recent years. This discovery is particularly noteworthy because since the 2000s, viruses of these lineages (including the globally prevalent seasonal H1N1 and the 2009 pandemic H1N1 virus) have developed widespread resistance to amantadine drugs in their M2 protein due to S31N and other site mutations. Therefore, the specific genetic background embedded in SD6591 itself is an important risk signal, which clearly indicates the direction that needs to be prioritized for subsequent functional validation. Although the main focus of this study is on comprehensive genomic feature identification and evolutionary tracing, the above findings have provided key genotype clues for further evaluating its potential drug sensitivity. Future monitoring and research can be based on this, by conducting targeted sequencing and functional characterization of the transmembrane region of M2 protein, to clearly determine whether it has similar resistance characteristics. If confirmed, such strains may have pre-existing resistance to some traditional antiviral treatment regimens, highlighting the need for continuous monitoring and clinical stockpiling of next-generation antiviral drugs.

We believe that these supplementary contents significantly enhance the depth of the paper discussion and better elucidate the potential biological and public health implications of genomic discoveries. Thank you sincerely for helping us improve our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

H1N1 swine influenza A viruses cause highly contagious respiratory diseases in pigs that are responsible for major losses to the farming industry.  Also, it has long been recognized that pigs as a sort of “mixing vessel”, based on their susceptibility to so many different viruses.  Indeed, the 2009 H1N1 influenza A pandemic was shown to a triple reassortant that originated in Mexico and spread rapidly worldwide due to its ability to cross species, including humans, birds and pigs.  All of this constitutes a clarion call for close monitoring of swine influenza A viruses.  With this in mind, the authors have mounted an in-depth characterization of two H1N1 influenza strains identified through routine surveillance of swine herds in Shandong, China.  This included viral gene sequencing, bioinformatic and phylogenetic analyses of multiple genes and recombination analyses.

The findings identified two strains of H1N1 swine flu.  Interestingly, one (called SD6591) appeared to be a unique triple-reassortant with the PB2, PB1, NP, NA, HA and NEP genes shared the highest homology with classical swine flu viruses.  The second isolate, SD6592, maintained genomic conservation with previously characterized H1N1 swine flu strains.  In addition, there is no evidence at all of intrasegment recombination events between the two strains.

This is considered a simple, straightforward accounting of two strains of H1N1 swine flu co-circulating in Shandong.  There are no detected weaknesses in the approach or interpretation of the data.  Although the study is considered complete and well-done, it’s contribution to the field is considered quite modest.  It does, however, serve to heighten our awareness of these viruses and add to our surveillance information that informs our prevention strategies.

 

 

 

 

Author Response

Sincerely thank you for reviewing our manuscript and providing a fair evaluation. We fully agree with your viewpoint that continuous monitoring of the swine influenza virus is crucial for public health. We also thank you for considering our research 'complete and well executed'. In response to your valuable feedback on enhancing the importance of research, we have made substantial revisions to the manuscript, emphasizing the potential warning significance and public health value of the research findings.

The specific modifications are as follows:

1. Strengthening research argument (Introduction section): In the introduction (lines 49-56), we have strengthened the discussion on the current monitoring data gap and the necessity of localized research, clarifying more clearly that this study aims to respond to the "urgent need for close monitoring" you mentioned and fill the cognitive gap in specific regions.
2. Deepen the discussion and highlight the importance of research findings:

For the unique recombinant strain SD6591: In our discussion (lines 309-332), we are no longer limited to describing its genotype, but focus on analyzing the rarity and potential risks of its combination of the PA gene and the M2 gene with drug resistance potential. We explicitly state that this strain can be considered as a "candidate" pre-epidemic strain with specific risk gene characteristics, and its appearance itself is an important warning signal.

For the conservative strain SD6592: We (lines 333-346) explained another layer of meaning for its high conservatism - that is, as a stable local virus gene pool, it may participate in future reassortment events, thereby amplifying the necessity of monitoring its co circulating partners (such as SD6591).

Through the above modifications, we aim to elevate this study from a "descriptive report" to a "monitoring analysis with warning value", aiming to clarify that even in seemingly routine monitoring, novel viruses with unique risk characteristics can be discovered, which is the core value of active monitoring. We believe that the revised manuscript can more fully reflect the contribution of this research to the field. Thank you sincerely for helping us improve our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reports on two interested swine H1n1 viruses that derived from probably triple-reassortment events. The authors did a good work in analyzing the evolutionary pathway and sequence similarity and genetic distance to other swine influenza viruses. The phylogenetic analysis is solid, which should be of good value to the swine influenza research community. The work can be improved in significance by characterizing the viral replication and transmission in various cell lines including human cells to further assess its zoonotic risk to humans.  

Author Response

Sincerely thank you for providing this very important and professional opinion. We fully agree that virus isolation is the gold standard for verifying virus activity and biological characteristics.

Firstly, we must honestly state that in the initial design and experimental stages of this study, our main focus was on high-throughput sequencing of preserved clinical samples to trace and identify viral genomes. Therefore, in the early stages of our research, we did not simultaneously carry out the work of isolating viruses from these specific samples. Later, considering the importance of the reviewer's comments, we also attempted to isolate the remaining samples from the virus. However, due to the prolonged storage time of the samples (over 5 years), we were ultimately unable to successfully isolate live viruses. We have identified this situation as a limitation of our study and provided a clear explanation in the discussion section of the paper (lines 372-388).

However, in order to directly and positively address your concerns about the functional significance of the virus, we conducted important supplementary experiments during the modification phase with a rigorous scientific attitude. In the Materials and Methods section (lines 105-132), it was added that a strain of H1N1 virus (named A/swine/China/Qingdao/2018 (H1N1)) was successfully isolated from same host and same region in 2018 and has been successfully published and introduced in the article [8]. This isolate has extremely high homology with the two strains (SD6591 and SD6592) obtained from sequencing in this study (see additional Tables 4 and 5), which provides us with a key breakthrough: we can use this highly homologous and known active virus to indirectly but strongly validate the biological characteristics of the virus represented by the sequencing results.

Based on the above analysis, we designed and completed in vitro replication kinetics experiments. We used H1N1 homologous isolates as the "functional representative strain" for our study sequence and infected the virus in PK-15, MDCK, and A549 cell lines (representing pig, dog, and human epithelial cells, respectively), with PR8 virus as the control. Select a time point (12 hours) and use Western blot at the protein level to detect the expression of viral proteins (such as NP proteins). Next, at the mRNA level, collect samples at 12 and 24 hours after virus infection and use fluorescence quantitative PCR (qRT PCR) to detect viral nucleic acid replication levels. Western blot is used to detect the expression of viral proteins, such as NP protein.  

The supplementary experimental results (newly added Figures 7 and 8) clearly indicate (please refer to lines 252-280 of the revised manuscript) that the H1N1 homologous isolate can be effectively replicated in all three cell lines. Firstly, virus replication ability was detected at the protein level in all three cell lines. Secondly, at the mRNA level, qRT PCR showed a significant increase in viral load over time, consistent with the replication trend of the PR8 control group. These data strongly indicate that viruses highly homologous to the strains we sequenced have active replication capabilities in various mammalian cells. Revise the conclusion (lines 390-413) to make it more relevant to the current research.

In summary, we cleverly compensated for the lack of direct virus isolation from this specific old sample by introducing and validating highly homologous isolated strains. This supplementary experiment not only directly responds to the comment, but more importantly, it elevates this study from a simple genomic analysis to a level related to viral function, greatly enhancing the persuasiveness of our research conclusion that the virus sequences identified in this study do indeed come from live viruses with replication ability, providing critical experimental support for their potential biological significance and cross species transmission risks. We have integrated the above new data and analysis into the corresponding chapters of the paper. Thank you sincerely for helping us improve our manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have conducted new experiments that addressed most of my concerns. There is no major issues noted during the review of this revised manuscript. From the data presented, these swine influenza viruses have the potential to replicate in human cells and cause influenza-like diseases in humans when new mutations are acquired.  

Author Response

Thank you for both recognizing the thoroughness of our revisions and supporting the publication of our study, and we truly value your expert insights and guidance throughout the review process.