Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.

The genome of hepatitis E virus (HEV) contains three open reading frames (ORF1-3), among which ORF2 encodes the viral capsid protein. It has been believed that ORF2 only encodes the structural protein for assembling the capsid of HEV virion. However, recent findings have revealed that during HEV replication, ORF2 also produces a substantial amount of glycosylated soluble protein secreted outside the host cells, known as the ORF2 secreted form (ORF2S). Although not involved in virion assembly, the soluble secreted form of ORF2 protein may play a crucial role in HEV infection and pathogenesis. This review presents an overview of research progress in HEV ORF2S.

ObjectiveTo analyze the evolutionary characteristics and genetic variations of the HA (hemagglutinin) and NA (neuraminidase) genes of influenza A(H1N1) viruses in Shanghai during 2024, to investigate their transmission patterns, and to evaluate their potential impact on vaccine effectiveness. MethodsFrom January to October 2024, throat swab specimens were collected from influenza like illness (ILI) patients at 4 hospitals in Shanghai. Real-time fluorescence ploymerase chain reaction (RT-PCR) was used for virus detection and isolation of H1N1 influenza viruses. Forty influenza A(H1N1) virus strains were sequenced using Illumina NovaSeq 6000 platform, followed by phylogenetic analyses, genetic distance analysis, and amino acid variation analyses of HA and NA genes. ResultsPhylogenetic tree of the HA and NA genes revealed that the 40 influenza A(H1N1) virus strains circulating in Shanghai in 2024 exhibited no significant geographic clustering, with a broad origin of strains and complex transmission chains. Genetic distance analyses demonstrated that the average intra-group genetic distances of HA and NA genes among the Shanghai strains were 0.005 1±0.000 6 and 0.004 6±0.000 6, respectively, which were comparable to or higher than those observed in global surveillance strains. Both HA and NA genes displayed frequent mutations. Compared to the 2023‒2024 and 2024‒2025 Northern Hemisphere A(H1N1) vaccine strains (WHO-recommended), the HA proteins of 40 Shanghai strains exhibited amino acid substitutions at positions 120, 137, 142, 169, 216, 223, 260, 277, 356 and 451, with critical mutations at positions 137 and 142 located within the Ca2 antigenic determinant. Furthermore, mutations in the NA protein were observed at positions 13, 50, 200, 257, 264, 339 and 382. ConclusionThe genetic background of the 2024 Shanghai influenza A(H1N1) virus strains is complex and diverse, and antigenic variation may affect vaccine effectiveness. Therefore, it is recommended to enhance genomic surveillance of influenza viruses, evaluate vaccine suitability, and implement more targeted prevention and control strategies against imported influenza viruses.

Objective To investigate the correlation of plasma homocysteine(Hcy)level and the polymorphisms of its key metabolic enzymes methylenetetrahy-drofolate reductase(MTHFR)gene with ischemic stroke(IS).Methods A total of 310 patients with IS were enrolled as the case group and 330 healthy subjects during the same period were selected as the control group.Plasma Hcy concentration was detected by enzyme cycling method,and the real-time fluorescent quantitative PCR(RT-qPCR)was used to detecte the genotypes of MTHFR C677T.Results The frequencies of TT genotype(36.13%),CT genotype(10.00%)and T allele(28.06%)of MTHFR gene C677T locus in stroke patients were significantly higher than that in control group(P＜0.05).The frequency of the TT genotype was significantly higher in IS group compared to control group,indicating a recessive mode of inheritance(P＜0.05);In the dominant mode of inheritance,the frequency of CT+TT genotype in IS group was also significantly higher than that in control group(P＜0.05);The plasma Hay concentration of MTHFR C677T genotype TT,CT and CC patients was statistically different(P＜0.05),which were(20.91±6.78)μmol/L(17.20±5.39)μmol/L,(14.35±4.32)μmol/L,respectively;The area under the ROC curve(AUC)of plas-ma total Hcy level was 0.610(95%CI:0.566～0.653,P＜0.001).It indicated that it might play an impor-tant role in predicting the risk of suffering from IS.Multivariate Logistic regression analysis showed that plasma Hcy level and MTHFR C677T gene polymorphism were important risk factors of IS.Conclusions Elevated plasma Hcy level is associated with IS,and the synergistic effect of elevated Hcy level and MTHFR C677T gene mutation may increase the risk of IS.

Objective To investigate the relationship between methylenetetra-hydrofolatereductase(MTHFR)gene polymorphism and serum homocysteine(Hcy)level and subtypes of ischemic stroke(IS).Methods The study was conducted according to the matched principle of case-control design,310 patients with IS and 330 healthy people during the same period were selected as the case group and the control group.Recycling enzyme method and the real-time fluorescent quantitative PCR(RT-qPCR)method were used to detect the level of serum Hcy and the genotypes of MTHFR C677T,respectively.Results There was a significantly difference in MTHFR C677T genotype and allele frequency between the case and control groups(P＜0.05).The correlation analysis with different subtypes indicated that the frequencies of CT genotype(38.02%),TT genotype(10.74%),and T allele(29.75%)were significantly different in the LAA group(OR=1.662,95%CI:1.058-2.608,P＜0.05;OR=2.373,95%CI:1.110-5.073,P＜0.05;OR=1.663,95%CI:1.190-2.323,P＜0.05);The frequencies of TT genotype(10.53%)and T allele(27.30%)in SAO group were also significantly different(OR=2.130,95%CI:1.046-4.336,P＜0.05;OR=1.474,95%CI:1.075-2.021,P＜0.05).Further analysis of serum Hcy level showed that LAA group(19.55±5.61)μmol/L and SAO group(16.37±5.20)μmol/L were significantly higher than the control group(14.46±4.61)μmol/L(P＜0.001);Among the patients of both subtypes the serum Hcy levels in those with CT genotypes and TT genotypes were significantly higher than those in patients of CC genotypes(P＜0.001).Conclusions The gene polymorphism of MTHFR C677T has a significant effect on Hcy level in pa-tients with LAA and SAO stroke.

Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.

The genome of hepatitis E virus (HEV) contains three open reading frames (ORF1-3), among which ORF2 encodes the viral capsid protein. It has been believed that ORF2 only encodes the structural protein for assembling the capsid of HEV virion. However, recent findings have revealed that during HEV replication, ORF2 also produces a substantial amount of glycosylated soluble protein secreted outside the host cells, known as the ORF2 secreted form (ORF2S). Although not involved in virion assembly, the soluble secreted form of ORF2 protein may play a crucial role in HEV infection and pathogenesis. This review presents an overview of research progress in HEV ORF2S.

Influenza viruses are common pathogens causing respiratory infections in humans. Among the four seasonal influenza viruses, influenza A virus H3N2 has become the leading cause of seasonal influenza illness and death, posing a great threat to public health and the economy. Since it first emerged and caused a pandemic in 1968, H3N2 has been circulating repeatedly in human beings and continually evades host immune attack by antigenic drift, resulting in a decrease in vaccine efficacy. In this paper, the antigenic evolution of influenza A virus H3N2, the impact of antigenic evolution on the selection of vaccine strains and some models for predicting the evolution of influenza viruses were analyzed and reviewed, which paved the road for understanding the antigenic evolution of influenza virus and vaccine development.

Influenza has caused high morbidity and mortality worldwide, seriously endangering human health and life. The continuous mutation of influenza virus has brought new challenges to the prevention and treatment of influenza. Animal models provide convenience for a comprehensive understanding of influenza virus pathogenesis, transmission mechanism, vaccine development, and evaluation of therapeutic effects. The construction and use of animal models of influenza virus infection vary in different studies, and the application of different animal models also has its own characteristics. This article reviewed the current status of the construction and use of various animal models, and summarized the advantages and limitations of animal models in evaluating the efficacy of antibodies, drugs and vaccines, with the aim of providing reference for the selection and optimization of animal models in the future.

Influenza viruses are responsible for a large number of infections and deaths annually, posing a serious threat to public health. Vaccination is the most effective measure to prevent influenza virus infection. However, current seasonal influenza vaccines only protect against closely matched circulating strains. Even with extensive surveillance and annual reformulation, yearly updated vaccines are still a step behind the fast-evolving viruses, often resulting in poor matches or less effective vaccines. Due to the relatively complex evolution of influenza A viruses, it is a new idea and a new means to prevent influenza epidemics by using a series of innovative technologies to develop universal influenza vaccines that can provide extensive and long-lasting protection against influenza viruses. This review summarized the latest progress in the development of universal vaccines targeting HA in the past three years, including design methods for universal vaccines targeting HA, HA stem and other conserved epitopes, compared the advantages and disadvantages of different technologies, explored the impact of immunization programs and strategies, and discussed the potential challenges to be overcome, hoping to provide reference for the successful development of universal vaccines.