1. Ongoing surveillance of the prevalence and mutation dynamics of avian influenza viruses has revealing the evolutionary origin of novel variants, and warning of potential outbreaks and public health risks.
1.1 Surveillance and Early Warning Network Establishment: A surveillance and early warning network has been established focusing on the important hosts of avian influenza viruses (AIV), including wild birds, poultry, and sentinel hospital patients (Science China-Life Sciences, Dec 2017; 60(12):1386-1391, first and corresponding author). This network conducts surveillance, early warning, and systematic research on novel and mutated avian influenza viruses.
1.2 Research on the Evolutionary Origin and Transmission Patterns of Novel AIVs
A comprehensive analysis of novel AIVs such as H7N9 and H5 reveals that in recent years, the HA and/or NA genes of these novel strains mostly originate from wild birds or waterfowl. These novel viruses or viral genes undergo reassortment with AIVs (e.g., H9N2) that are endemic in poultry through direct or indirect contact. This reassortment results in the formation of new reassortant AIVs. The HA/NA genes of these reassortant viruses come from wild bird AIVs, while the internal gene segments are from poultry AIVs. This genetic combination enables reassortant viruses with novel HA/NA genes to adapt, replicate, and survive in poultry. After the formation of novel AIVs, they are spread across the country through live poultry trade and transportation. Different AIVs are further mixed and reassorted as they are transported with live poultry to various markets, leading to continuous reassorting of internal genes from AIVs of different regions and lineages (e.g., H7N9’s internal genes continue to reassort with H9N2 viruses). Human infections with novel AIVs typically occur through contact with infected poultry or virus-contaminated environments, such as live poultry markets. Therefore, during the origin, evolution, and spread of novel AIVs, wild birds act as a “gene pool”, live poultry markets serve as “incubators or mixers” for viral mutations, live poultry trade and transportation act as vehicles for transmission (with wild birds also acting as intermediaries or carriers in some novel AIV transmissions), and the widely circulating poultry AIVs (e.g., H9N2) function as a universal “engine or grafting rootstock.” These findings were published in Journal of Virology, 2015, 89(17): 8671-8676; Emerging Infectious Diseases, 2014, 20(12): 2076-2079; Emerging Infectious Diseases, 2017, 23(4): 637-641; Emerging Microbes Infections, 2016, 5, e125; Virologica Sinica, 2016, 31(4): 300-305; and Scientific Reports, 2015, 5: 12986.
1.3 H7N9 AIV Genetic Evolution and Early Warning Studies
In 2015, an early warning was issued by CASCIRE, highlighting the trend of increased pathogenicity in the H7N9 virus. This prompted the deployment of monitoring tasks, with close attention to mutations in the HA cleavage site region. A large number of H7N9 viruses were isolated and identified from healthy chickens and ducks, as well as from sick poultry and human cases. Between December 2017 and January 2018, two cases of human infection with highly pathogenic H7N9 (HP-H7N9) avian influenza viruses (HPAIV) were identified and reported (Journal of Infection, 2017, 75(1): 71-75), and during the same period, HP-H7N9 viruses were also detected in poultry. A systematic study was conducted on the evolutionary variations and new characteristics of the 5th wave H7N9 virus based on previous data on its distribution and genomic information (Journal of Virology, 2018, 14;92(11):e00301-18). In addition, collaborative monitoring with CASCIRE partners was used to study the infectivity of identified HP-H7N9 strains from poultry and humans using SPF chickens and mice (Journal of Virology, 2018, 92(2): pii: e00921-17).
In February 2017, the Chinese Center for Disease Control and Prevention and the Ministry of Agriculture confirmed the emergence of HP-H7N9 virus and reported it to the World Health Organization (WHO) and the World Organization for Animal Health (OIE). The early warning of enhanced pathogenicity in H7N9 mutations was verified. We promptly reported our research progress and prevention strategies to the Chinese Academy of Sciences,and our report was adopted by the General Office of the Communist Party of China. Genetic analysis revealed that some HP-H7N9 viruses isolated from poultry and the environment carried mutations associated with resistance to neuraminidase inhibitors (NAIs) such as oseltamivir, as well as mammalian adaptation mutations. Further analysis showed that, compared to LP-H7N9, the HP-H7N9 virus had specific mutation sites in the HA and NA genes, with a diverse composition of amino acid residues.
Based on the diversity of amino acid composition at the HA cleavage site and the mutation sites associated with NAI resistance, a nucleic acid detection method was designed to simultaneously differentiate high-pathogenicity and low-pathogenicity H7N9 viruses, as well as resistant and non-resistant (NAI) strains (Patent Number CN201710804952.2 and CN201710264272.6). This method has been applied to the study of H7N9 mutation and early warning.
1.4 The Predominant Prevalence of H5N6 AIV and Its Genetic Origin and Evolutionary Mechanisms:
1.4.1 Discovery of the Predominant Prevalence of H5N6: In the northern regions of China, H9N2 has been identified as the predominant avian influenza virus (AIV) in poultry. In contrast, the Yangtze River Delta, Central China, and South China exhibit a notable presence of H7N9. However, in areas south of the Yangtze River Delta, the prevalence of H5N6 has significantly increased, gradually supplanting H5N1 as the dominant circulating strain.
1.4.2 Transmission of H5N6 and Identification of Its Host Carriers: Research has demonstrated that H5N6 and H6N6 are the predominant viral subtypes circulating in ducks, while H9N2 is the primary subtype in chickens. Ducks have been identified as a key player in the generation and spread of H5N6 viruses. Additionally, instances of wild birds and feral cats being infected and serving as carriers of the H5N6 virus have been documented.
1.4.3 Revelation of the Origin and Evolutionary Mechanisms of H5N6: H5N6 originated from the reassortment of H5Ny and H6N6. The combination patterns of the H5 and N6 genes exhibit lineage-specific evolutionary characteristics. During the virus's prevalence and transmission, its internal genes underwent continuous reassortment with low pathogenic avian influenza viruses (LPAIVs), resulting in the formation of at least 34 distinct genotypes. Through natural selection, four dominant genotypes (G1, G2, G1.1, and G1.2) have emerged. G1 and G2 are the earliest formed genotypes. G1.1 evolved from G1 through reassortment with the PB2 gene from an H6 virus, while the internal genes of G1.2 are derived from H9N2/H7N9.
1.4.4 Tracing the Origin and Identifying Genetic Characteristics of H5N6 Viruses Involved in Cross-Species Human Infections: It has been determined that the four dominant genotypes mentioned above are responsible for the known human cases of infection. Among these, the G1.2 genotype shares a similar genetic composition with H7N9 and H10N8 viruses and has caused at least five human infections. Source tracing revealed that humans are primarily infected through direct contact with virus-carrying poultry or contaminated materials, with no evidence of sustained human-to-human transmission to date.
1.4.5 H5N6 Epidemic Spread Warning: Based on previous research findings, an early warning was issued regarding the potential spread of H5N6 via migratory wild birds. At the end of 2016, outbreaks of H5N6 occurred among domestic poultry and wild birds in South Korea, and to date, H5N6 has been successively detected in South Korea, Japan, Greece, the Netherlands, the United Kingdom and other regions.
The related research findings were published in Cell Host & Microbe, 2016, 20(6): 810-821 (cover article), Scientific Reports, 2016, 6: 29888, and Journal of General Virology, 2015, 96: 975-981. These studies have provided a scientific basis for the prevention and control of avian influenza, offering practical guidance. The research has been reported and commented on by outlets such as Science and Technology Daily. Moreover, some of these findings were recognized as leading-edge research in the "Comparison of Sino-American Research Capabilities: Analysis Based on 'Frontier Research in 2016'" under the category “Identification and Characterization of Novel Recombinant Avian Influenza Viruses (H5N8 and H5N6). The research is at the forefront of the field.
1.5 Discovery of the Shift in Epidemic Dynamics of Avian Influenza Viruses:
After 2016, H9N2 AIV, which had mainly been circulating in chicken populations, gradually became absolutely dominant in both chicken and duck populations. H5N6 completely replaced H5N1 in poultry circulation and is showing a trend of antigenic variation. Since 2018, H7N9 AIV has gradually disappeared in poultry, with only occasional detections. However, a novel variant of recombinant highly pathogenic avian influenza virus (HPAIV), H7N3, was identified. In this variant, the HA gene originated from H7N9 HPAIV, the NA gene from an HxN3 low pathogenic avian influenza virus (LPAIV), and the internal genes from LPAIV. Several novel viruses were also identified, including H9N9 (a recombinant product of H9N2 and H7N9 viruses), H9N6 (a recombinant product of H9N2 and HxN6 viruses), H10N3, and H10N8 (which arose from recombination between H10Ny and HxN3 or HxN8 viruses; the internal genes originated from LPAIV, and the HA gene of H10N3 is closely related to that of the virus that infected humans in 2020, though it differs somewhat from the H10N8 virus that infected humans in 2013). These findings revealed the genetic diversity of AIV carried by poultry and elucidated the genomic evolutionary mechanisms of AIV subtypes such as H9N2, H5N6, H7Ny, H6Ny and H10N3. For the first time globally, a cluster infection event in domestic cats and humans caused by the cross-species transmission of H9N2 AI was identified, noting that patients exhibited atypical flu-like symptoms. It was discovered that nearly all circulating H9N2 strains, along with some H7N9, H6N2, H7N3 and H10Ny strains, have a preference for or possess the ability to bind to human-type receptors (α2-6-SA), indicating an increased risk of these circulating AIV infecting humans.
Monitoring and genetic variation studies of viruses carried by migratory birds have revealed that the H5N8 and H5N1 AIVs of the Clade 2.3.4.4b branch continuously mutate during the migration of wild birds. These viruses reassort with various low-pathogenic avian influenza viruses (LPAIVs) to form multiple new genotypes, which are then carried into China by migratory birds, causing illness and death among wild bird populations. The study identified and named two new evolutionary branches of H5 viruses, namely Clade 2.3.4.4b.1 and Clade 2.3.4.4b.2, and three sub-branches within Clade 2.3.4.4b.2. It was found that the newly identified H5N1 and H5N8 viruses from wild birds exhibit antigenic drift compared to vaccine strains. These viruses have already triggered outbreaks of avian influenza in wild birds and/or poultry across Europe, Asia, Africa, and the Americas, as well as human infection cases, posing a threat to the poultry industry and human health.
Comparative analysis revealed that the AIV positive rate in live poultry markets decreased significantly from 26.90% during 2014-2016 (Cell Host & Microbe, 2016 Dec 14;20(6):810-821) to 12.73% during 2016-2019. Concurrently, human cases of H7N9 virus infection gradually disappeared. This suggests that China's comprehensive prevention and control measures, including vaccination and management of live poultry markets, have played a positive role in controlling avian influenza among poultry and reducing human infections. However, continuous monitoring of AIV mutations and epidemic trends remains necessary to prevent outbreaks of novel and variant strains in both poultry and humans.
Academic Value and Scientific Significance:
These studies elucidate the dynamics of AIV epidemics and variations in China, providing early warnings about the potential threats posed by novel and variant viruses such as H5N1, H7N3, H5N6, and H9N2 to poultry and human health. For the first time, we identify and explore the reasons behind the cross-species transmission and familial cluster infections of the H9N2 virus among "poultry, cats, and humans," enriching our understanding of the genetic and pathogenic characteristics of the H9N2 virus and its transmission ecological. The study highlights the high public health risk associated with H9N2 AIV and calls for global attention and enhanced prevention and control measures. Moreover, the newly identified H5N6 strain, previously flagged as a concern, has been confirmed by international research teams (PLoS Pathogens, 2021,17(7):e1009381 and Antiviral Research, 2020, 182:104886) to exhibit an increased risk of cross-species transmission and enhanced pathogenicity following drug-resistance mutations. The research findings have been published as the first or corresponding author in the following journals: Nature Communications, 2020 Nov 20;11(1):5909, Lancet Microbe, 2022, 3(11):e804-e805, Emerging Infectious Diseases, 2023 Jun, 29(6):1244-1249, Emerging Microbes & Infections, 2023 Dec;12(1):2143282, Emerging Microbes & Infections, 2021, 10(1):1819-1823, Transboundary and Emerging Disease, 2019, 66(1):592-598. These publications provide scientific evidence to support the formulation of avian influenza prevention and control strategies, as well as the development and application of diagnostic methods for detecting variant viruses.
2. Research on the Pathogenic Mechanisms of Influenza Virus
Based on pathogen surveillance, genetic variation, and early warning analysis, cell and animal infection models were established for novel avian influenza viruses (AIVs) to investigate the mechanisms of AIV pathogenicity and cross-species transmission to mammals. By elucidating these pathogenic mechanisms, antiviral drugs were designed, and their molecular mechanisms of action were studied.
2.1 Identification of Virulence Genes in H7N9 Virus Infecting Mammals
The H7N9 AIV, which emerged in 2013, exhibited low pathogenicity in poultry, with infected chickens showing no typical clinical symptoms. However, it demonstrated high pathogenicity in humans. It is rare for a low-pathogenicity avian influenza virus to exhibit such high pathogenicity in humans. Genetic evolutionary analysis revealed that the internal genes of the H7N9 virus originated from the H9N2 virus. However, infections with the H9N2 virus in humans usually cause only mild cold-like symptoms, and confirmed cases remain rare. This suggests that the internal genes of the H7N9 virus have undergone mammalian-adaptive mutations. Moreover, previous studies have demonstrated that the internal genes, particularly the polymerase genes, play a critical role in viral adaptation to new hosts. Therefore, leveraging the low pathogenicity of H9N2 virus in mammals, a strain of H9N2 virus was selected as the genetic background. Using established cell and mouse infection models, the impact of each internal gene of H7N9 on the virus's ability to infect mammals and other biological functions was systematically evaluated. The study identified that the PB2, NP, and M genes of the H7N9 virus determine its pathogenicity in mammals. These three internal genes, together with the surface HA and NA genes, contribute to the virus's ability to infect human cells. The PB2 E627K mutation was identified as a key virulence determinant of H7N9 virus infection in mammals. Furthermore, studies demonstrated that avian-origin H7N9 viruses could acquire host-adaptive mutations at position 627 of PB2 within four days of infecting mammals, enabling rapid cross-species transmission from birds to mammals. This significantly enhanced the virus's virulence in mammals, leading to host mortality. This research provides a theoretical foundation for the development of AIV antiviral drugs and influenza epidemic prevention and control. The findings were published as first author in the Journal of Virology, 2015, 89(1):2-13, and garnered widespread attention, coverage, and commentary from the scientific community and the public.
2.2 Interpretation of the Correlation between NA Stalk Length and H7N9 Virus Infection and Pathogenicity
Analysis revealed that the H7N9 virus naturally possesses a deletion of five amino acids at positions 69-73 in the NA stalk. NA, which exhibits neuraminidase activity, plays a crucial role in the later stages of viral replication. The deletion in the NA stalk is considered a molecular marker for the adaptation of wild waterfowl AIV to terrestrial poultry, and it may also enhance the pathogenicity of AIV in mammals. Concurrently, preliminary research by the applicant demonstrated that the H7N9 virus's HA, NA, and virulence genes PB2, NP, and M collectively contribute to the virus's ability to infect human cells. Consequently, utilizing established cell, SPF chicken, and mouse infection models, functional studies were conducted on the NA stalk. The results indicated that the deletion of these five amino acids does not affect the virus's enzymatic activity, replication capability in vitro and in vivo, or pathogenicity in SPF chickens and mice. However, as the virus continues to spread and evolve, longer deletions in the NA stalk, such as the 19-20 amino acid deletions observed in H5N1, may emerge. Therefore, the applicant conducted preemptive research to investigate the impact of longer NA stalk deletions (19-20 amino acids) on H7N9 virus infection and pathogenicity. The findings showed that while longer deletions in the NA stalk (19-20 amino acids) do not affect the virus's pathogenicity in SPF chickens, they significantly increase its pathogenicity in mice. Some of these research findings were published in Journal of Virology (2016, 90(4):2142-2149) as a first and corresponding author, providing a scientific basis for H7N9 virus surveillance and risk assessment.
2.3 Elucidation of the Molecular Mechanisms Underlying H7N9 Virus Resistance to NAIs
Genetic variation analysis revealed that the NA gene of the H7N9 virus comprises two distinct evolutionary branches, represented by AH/1/13(H7N9) and SH/1/13(H7N9), respectively. Further analysis identified a difference at position 294 (marked as position 292 in the N2 reference sequence) in the NA of AH/1/13 and SH/1/13, where the amino acids R and K are present, respectively. This site is located within the enzymatic active site region. It was hypothesized that the R294K mutation might confer neuraminidase inhibitor (NAI) resistance to the H7N9 virus. If such H7N9 viruses were to develop resistance to NAIs, it would pose significant challenges to clinical treatment. The applicant's research on this issue confirmed that SH/1/13-like viruses carrying the R294K mutation exhibited significant resistance to NAIs, particularly showing a reduction in sensitivity to Oseltamivir (Tamiflu) by more than 100,000-fold, with IC50 values increasing to the μM level. In-depth studies revealed that the K294 mutation caused a 1.5 Å deviation between N9 and the carboxyl end of NAIs, along with the insertion of two water molecules between K294 and NAIs, weakening the binding between N9 and NAIs, thereby leading to drug resistance. Utilizing established cell infection models, it was determined that SH/1/13-like viruses exhibited low-level replication capability in vitro, suggesting that these viruses are not yet fit for survival. Meanwhile, although the sensitivity of SH/1/13-like viruses to other NAIs such as Zanamivir, Peramivir, and Laninamivir decreased by 20-500 times, the IC50 values remained at the nM level. Therefore, the current clinical treatment for H7N9 can reasonably use NAIs without excessive concern over the emergence of resistant strains. This research elucidated the molecular mechanisms of H7N9 virus resistance to NAIs at the protein, virus, and structural levels, providing guidance for the clinical treatment of H7N9. The study was published as a co-first author in Cell Research, 2013, 23(12):1347-1355 (cover article). Cell Research published an article titled "Solving the mystery of H7N9 by crystal balls," commenting on this research.
2.4 Antiviral Drug Design Based on Resistance Mechanisms and Its Molecular Mechanisms of Action
Building on the elucidation of the molecular mechanisms underlying H7N9 virus resistance to neuraminidase inhibitors (NAIs), and based on the tetrameric structure of NA and the spatial binding site of Zanamivir on the NA tetramer, polyethylene glycol (PEG)—a biocompatible linker—was utilized to connect four Zanamivir molecules without disrupting its pharmacophore, thereby constructing Tetravalent Zanamivir. Theoretically, Tetravalent Zanamivir can bind to the NA tetramer in a 1:1 ratio. Crystallographic studies confirmed this design concept, demonstrating that the complex formed between Tetravalent Zanamivir and the NA tetramer is thermodynamically more stable, enhancing the effective concentration of Zanamivir and enabling robust inhibition of both resistant and non-resistant H7N9 viruses. Furthermore, Tetravalent Zanamivir exhibited potent inhibitory effects against newly emerged highly pathogenic (HP) H7N9 resistant viruses. Additionally, pharmacokinetic studies revealed that the half-life of Tetravalent Zanamivir in rats (intravenous injection) was 2.25 times longer than that of monomeric Zanamivir. Partial research findings were published in the Journal of Medicinal Chemistry, 2016, 59(13):6303-6312 (Patent Authorization: ZL201410068706.1).
Previous studies confirmed that PB2 is a key virulence gene for H7N9 infection in mammals, and Ribavirin exerts its antiviral effects primarily by inhibiting polymerase. Therefore, in the absence of new drugs, laboratory evaluations were conducted to assess the efficacy of Ribavirin against both resistant and non-resistant H7N9 viruses. The results demonstrated that Ribavirin exhibits anti-H7N9 activity both in vitro and in vivo (Protein Cell, 2016, 7(8):611-614). Leveraging the molecular mechanisms of Zanamivir and Ribavirin in combating influenza viruses, a novel compound drug "Z-R" was designed and synthesized. The design rationale for Z-R is its dual action on both viral polymerase and NA.
2.5 Infection and Pathogenesis Mechanisms of Cross-Species Transmission of H7N9, H5N6, and H5N1 AIV
In the study of avian influenza viruses (AIV), it has been observed that the number of infection cases during the fifth wave of the H7N9 outbreak has significantly increased. Has the H7N9 AIV mutated, leading to an enhanced ability for cross-species transmission from birds to humans? Concurrently, it has been found that HP-H7N9 (H7N9 HPAIV) has caused a case fatality rate of approximately 50% among infected patients, significantly higher than the 39% case fatality rate caused by LP-H7N9 (H7N9 LPAIV). Moreover, the 65.2% case fatality rate resulting from cross-species infection of humans by H5N6 is substantially higher than the 50% and 39.2% case fatality rates caused by H5N1 and H7N9 infections, respectively, and the <0.2% case fatality rate caused by the 2009 H1N1 pandemic influenza virus (pdmH1N1, a reassortant of avian, swine, and human influenza viruses). The infection and pathogenesis mechanisms of cross-species transmission of H5N6, H5N1, and H7N9 AIVs represent significant scientific questions.
Mechanisms of Pathogenicity Induced by AIV Infection: Compared to pdmH1N1, H5N6, H7N9, and H5N1 AIVs induce a pronounced "cytokine storm" in the host post-infection, with H5N6 AIV particularly eliciting the highest levels of cytokines (IL-6, IL-10, IL-12p40, MIG, IFN-α2, MCP-3, IP-10, TRAIL, etc.). Furthermore, it was observed that cytokine levels in the serum of recovered cases from H5N6 AIV infection rapidly decreased after infection, whereas in fatal cases, these levels remained persistently high. Neutralizing antibodies were detected at certain levels in both fatal and recovered cases. Recovered patients from H5N6 infection began to exhibit virus-specific T cell responses from the 10th day after the onset of clinical symptoms, showing a gradually increasing trend, which was not detected in fatal cases. The findings suggest that the "cytokine storm" induced by AIV in the host is a direct cause of the significantly higher mortality rate compared to pdmH1N1 infection; moreover, virus-specific T cell immune responses are closely related to the recovery of infected patients. In-depth research reveals that the NS1 protein of AIVs such as H5N6 binds to the host protein SSU72, leading to the ubiquitination and degradation of SSU72, which triggers transcriptional read-through of the complementary strand of the STAT gene, thereby inhibiting the expression of downstream antiviral genes. This results in the evasion of host immunity by AIV, leading to host cell infection, death, and disease exacerbation. The results indicate that the interaction of AIV with the host SSU72 protein, thereby inhibiting the host's innate antiviral immune response, is the immunological mechanism underlying the significantly higher pathogenicity of H5N6, H7N9, and H5N1 AIV infections compared to pdmH1N1.
Variation and Pathogenic Mechanisms of H7N9 AIV: During the fifth epidemic wave, H7N9 AIV exhibited a broader distribution, concurrently circulating in live poultry markets and farms. The prevalent viruses demonstrated diversity in receptor binding affinity, pathogenicity and replication capacity in poultry (chickens and ducks), mice, and ferrets, as well as in their horizontal transmission capability among ferrets. The pathogenicity and transmission capacity in mammals did not surpass that of the progenitor virus A/Anhui/1/2013(H7N9). However, it was discovered that, apart from human-origin strains, avian-origin H7N9 strains widely carried mammalian-adaptive genetic mutations, and some avian-origin strains exhibited oseltamivir resistance mutations in the NA gene. This evidence suggests that the transmission capability of H7N9 AIV has not increased; the widespread distribution of H7N9 viruses carrying mammalian-adaptive mutations contributed to the outbreak of the fifth epidemic wave. Additionally, it was found that the pulmonary pathological damage induced by HP-H7N9 and LP-H7N9 infections was similar, with no significant differences in the levels of cytokines induced in the host or in viral replication and shedding in target organs. However, following HP-H7N9 infection, the rate of oseltamivir resistance mutations in the NA gene (80.0%) was significantly higher than that in LP-H7N9 (14.3%), indicating that the antiviral efficacy of drugs diminishes with the emergence of resistance mutations, thereby increasing the mortality rate among infected patients. The increased rate of resistance mutations to oseltamivir and other drugs in the NA gene constitutes the molecular basis for the significantly higher lethality of HP-H7N9 compared to LP-H7N9.
Academic Value and Scientific Significance:
This study elucidates the pathogenic mechanisms of cross-species infection by H5N6, H7N9, and H5N1 AIVs from both viral and host immune perspectives, enriching the understanding of the mechanisms underlying cross-species transmission of AIVs. The research proposes that controlling drug resistance mutations during viral infection, enhancing virus-specific T cell immune responses, and reducing the interaction between the virus and the host SSU72 protein are of significant importance for the prevention and control of cross-species transmission of AIVs. Based on the research progress, recommendations for AIV prevention and control were adopted by the General Office of the Communist Party of China. The findings have been published in the following journals: Clinical Infectious Diseases, 2019 Mar 19;68(7):1100-1109; Cellular & Molecular Immunology, 2022 Jun;19(6):702-714; Journal of Infection, 2019, 78(3):241-248; Emerging Microbes & Infections, 2019, 8(1):94-102; and Chinese Journal of Virology, 2023, 39(04):949-961.
Study on the Mutation and Transmission Characteristics of Seasonal Influenza Viruses
3.1 Elucidating the Respiratory Pathogen and Influenza Virus Transmission Characteristics During the First Influenza Season After the COVID-19 Pandemic
In the first winter following the COVID-19 pandemic, there was a significant increase in acute respiratory infections caused by various respiratory pathogens, leading to a surge in the number of patients with fever and hospitalizations, which resulted in a significant public health burden. The increase in respiratory infections in certain age groups, such as delayed Mycoplasma pneumonia in children, deviated from the usual seasonal trends. All these phenomena could be attributed to the immune gaps that accumulated during the COVID-19 pandemic.
Since mid-November, extensive surveillance and early warning studies for respiratory pathogens were initiated. The study included 1,507 patients of all ages with respiratory symptoms who visited fever clinics, consisting of 765 males and 742 females. In terms of age distribution, 158 were children and 1,349 were adults. Advanced real-time fluorescent PCR technology was used to precisely detect 27 common respiratory pathogens, including various bacteria and viruses.
Overall monitoring revealed a significant change in the epidemiological trends of respiratory pathogens in North China following the COVID-19 pandemic. In contrast to the pandemic period, the current trend is characterized by the co-circulation of multiple pathogens, with different pathogens alternately dominating during different epidemic phases. Three distinct phases were observed based on the prevalence trends of the dominant pathogens.
Phase 1 (Nov-Dec 2023): The first phase, from November to the end of December 2023, was dominated by Influenza A Virus (IAV). During this period, the detection rate of SARS-CoV-2 was only 5.1%, significantly lower than during the pandemic, indicating that the epidemic had been effectively controlled.
Phase 2 (Jan 2024): The second phase, starting in early January 2024, saw Influenza B Virus (IBV) becoming the dominant pathogen, with a rapid rise in detection rates, reaching 29.8%. However, this trend weakened in early February, possibly due to the establishment of immunity and the implementation of other control measures.
Phase 3 (Feb 2024): By mid-February 2024, SARS-CoV-2 detection began to rise, signaling the start of the third phase. By mid-March 2024, SARS-CoV-2 detection reached its peak at 15.8%. SARS-CoV-2 detection rates gradually declined in April, indicating a temporary retreat in the virus’ prevalence.
Among the 1,507 cases of respiratory pathogen infections, 284 (18.8%) had co-infections with two or more pathogens. Notably, co-infections with Haemophilus influenzae and other viral and bacterial pathogens were particularly prominent, occurring in 66 cases (4.4%), followed by IBV (64 cases, 4.2%) and IAV (40 cases, 2.7%). It is noteworthy that 18 co-infections (1.2%) involved two viruses, including 13 cases of IAV/IBV-HBoV and 5 cases of HBoV/HPIV-I. Correlation analysis further revealed potential interactions between these pathogens. Specifically, there was a significant negative correlation between SARS-CoV-2 and influenza viruses, especially with IBV (P < 0.0001). Additionally, Haemophilus influenzae co-infection was more likely with other pediatric pathogens such as Adenovirus (ADV) (P < 0.05), Human Metapneumovirus (HMPV) (P < 0.05), and Human Rhinovirus (HRV) (P < 0.0001), but showed a significant negative correlation with SARS-CoV-2 (P < 0.0001). On the other hand, Moraxella catarrhalis exhibited a positive correlation with coronaviruses, including SARS-CoV-2 (P < 0.0001) and HCoV-229E (P < 0.0001). Furthermore, there was a significant positive correlation between Human bocavirus (HBoV) and Human Parainfluenza Virus Type 1 (HPIV-I). In contrast, co-infection between influenza viruses and other respiratory viruses was relatively rare.
Additionally, a restricted cubic spline (RCS) analysis was used to examine the relationship between respiratory pathogen infections and age in the collected samples. The infection risk profiles for SARS-CoV-2, IAV, and IBV showed similar patterns: susceptibility gradually increased in patients under 30 years, peaked in the 30-40 age group, and then declined. SARS-CoV-2 and IAV primarily infected middle-aged and elderly individuals, while IBV mainly affected adults, with a sharp decrease in infection risk after the age of 40. In contrast, Haemophilus influenzae showed higher susceptibility in adolescents, with a substantial decrease in risk as age increased, stabilizing in older adults. HBoV, RSV, Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, and Staphylococcus aureus displayed a U-shaped relationship with age. In the population under 30, the risk of infection with these pathogens decreased with age, reaching its lowest point in adolescents and middle-aged individuals, but risk increased again with age in older adults. For Streptococcus pneumoniae and other pathogens, the lack of a significant nonlinear age-related susceptibility pattern may be due to limited sample size or an inherent lack of strong age-dependent susceptibility patterns for these specific pathogens in the study population.
3.2 Genetic Characteristics of H1N1 Influenza Virus After the COVID-19 Pandemic
During the COVID-19 pandemic, the public health measures implemented to mitigate the spread of SARS-CoV-2 also significantly hindered the global transmission of influenza viruses. Following the optimization of COVID-19 control policies, global influenza activity began to rebound. The A(H1N1)pdm09 strain, known for its high transmissibility, emerged in a new wave of outbreaks in China in February 2023, with a dramatic increase in cases. To explore the genetic characteristics of the H1N1 influenza virus post-policy optimization, this study conducted a genetic and phylogenetic analysis of 48 strains collected from Hunan and Jiangsu provinces. Phylogenetic trees of the HA gene and antigenic site mutations revealed a mismatch between the circulating H1N1 strains in China and the WHO-recommended vaccine strains for the same or subsequent periods. This indicates a need for a more precise and personalized vaccine updating strategy. The results of this study were published in Virologica Sinica, 2024, S1995-820X(24)00071-3.
3.3 Genetic Variability and Biological Characteristics of H3N2 Influenza Virus During the COVID-19 Pandemic
During the COVID-19 pandemic, overall influenza virus activity declined, yet the H3N2 subtype caused three distinct outbreaks of influenza. Therefore, it is essential to understand the genetic variation and biological characteristics of the H3N2 influenza virus during this period.
Between late 2019 and the summer of 2022, a total of 579 samples were collected from patients exhibiting influenza-like symptoms. Of these, 499 samples were collected in Beijing from late 2019 to early 2020 (from hospitals that pre-screened positive for influenza virus), with 404 (80.96%) testing positive for H3 by qRT-PCR. Additionally, 80 samples were collected in Hainan in the summer of 2022, 18 (22.5%) of which were H3-positive. Using next-generation sequencing (NGS), full-genome sequences were obtained from 36 H3N2 influenza viruses.
Phylogenetic analysis demonstrated that the strains circulating in China from late 2019 to early 2020 and in the summer of 2022 belonged to distinct HA branches, with both showing genetic divergence from the vaccine strains used during the same period. The phylogenetic tree of the NA gene showed a similar topology to that of the HA gene. The H3N2 viruses from Beijing were divided into two main evolutionary branches: 10 strains clustered with the next flu season’s vaccine strain (HK/2019) and strains circulating in North America and Asia, while 9 other strains formed a separate branch represented by A/Beijing/CAS-CJFH77-1222/2019 (BJ77/19-like). Notably, the strain A/Beijing/CAS-CJFH99/1225/2019, which was related to the vaccine strain HK/2019 in terms of its HA gene (belonging to the 3C.2a1b.1b sublineage), had an NA gene belonging to the BJ77/19-like lineage, suggesting genetic reassortment between different HA and NA lineages of H3N2 viruses.
The 17 H3N2 strains from Hainan clustered with the vaccine strain A/Cambodia/e0826360/2020, which was used in the 2021-2022 flu season, but showed a notable genetic divergence. Furthermore, hemagglutination inhibition (HI) and microneutralization (MN) assays revealed antigenic differences between the vaccine strains and the circulating H3N2 strains.
To evaluate the pathogenic properties of H3N2 influenza viruses, representative strains were selected for in vitro and in vivo replication and pathogenicity assays. The results indicated that H3N2 strains were capable of replicating in MDCK cells, although the replication efficiency varied between strains. All strains exhibited better replication at 37°C than at 33°C.
In vivo studies using mouse and guinea pig models revealed that the H3N2 strains could replicate effectively in the respiratory tracts of mice and the nasal cavities of guinea pigs without prior adaptation.
These findings highlight the genetic mismatch between the H3N2 strains circulating during the COVID-19 pandemic in China and the WHO-recommended vaccine strains, underscoring the need for enhanced international collaboration, the development of universal influenza vaccines, and strengthened epidemiological surveillance to mitigate future pandemics. The research results have been accepted for publication in hLife.
Academic Value and Scientific Significance:
This study provides critical insights into the genetic variations of H1N1 and H3N2 influenza viruses before and after the COVID-19 pandemic, as well as the evolving trends of respiratory pathogens in North China. It elucidates shifts in pathogen dynamics and potential interactions between different pathogens, highlighting the varying susceptibility to infections across different age groups. These findings are of significant importance in developing targeted prevention and treatment strategies, assisting public health authorities in better responding to respiratory disease outbreaks and reducing the occurrence and transmission of respiratory diseases. The results of this research have been published in China CDC Weekly (2025 Jan; 7(4):113-120) and Virologica Sinica (2024, S1995-820X(24)00071-3) and have been accepted for publication in hLife