Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Phylogenetic and epidemiological characteristics of H9N2 avian influenza viruses in Shandong Province, China from 2019 to 2021

H9N2 avian influenza virus(AIV) has widely circulated in poultry worldwide and sporadic infections in humans and mammals. During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2AIVs. Phylogenetic analyses showed that the eight gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades(HA and NA genes), G1-like clades(PB2 and M genes), and SH/F/98-like clades(PB1, PA, NP and NS genes). The isolates showed mutation sites that preferentially bind to humanlike receptors(HA) and mammalian fitness sites(PB2, PB1 and PA), as well as mutations in antigen and drug resistance sites. Moreover, studies with mice revealed four isolates with varying levels of pathogenicity. The average antibody titer of the H9N2 AIVs was 8.60 log2. Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

Labeling of influenza viruses with synthetic fluorescent and biotin-labeled lipids

Direct labeling of virus particles is a powerful tool for the visualization of virus–cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid(FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fluorescent(fluo-Ad-DOPE) or biotin-labeled(biot-CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin(HA) receptor specificity, neuraminidase(NA) activity, or replicative ability in vitro. Our data clearly show that neither construct significantly affected influenza virus infectivity or viral affinity to sialyl receptors. Neither construct influenced the NA activities of the influenza viruses tested, except the A/Puerto Rico/8/34(H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze influenza virus infection in vitro.

A duplex RT-PCR assay for detection of H9 subtype avian influenza viruses and infectious bronchitis viruses

H9 s ubtype avian influenza virus（AIV） and infectious bronchitis virus（IBV） are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction（RT-PCR） assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR（d RT-PCR） was established. Two primer sets target the hemagglutinin（HA） gene of H9 AIV and the nucleocapsid（N） gene of IBV, respectively. Spec ific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the d RT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×10^1, 1.5×10^1 and 1.5×10^1 50% egg infective doses（EID_（50）） m L^–1, respectively. The concordance rates between the d RT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the d RT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and survei llance of H9 AIVs and IBVs.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

Strengthened Monitoring of H5 Avian Influenza Viruses in External Environment in Hubei, 2018

The contamination status of H5 avian influenza viruses and distribution of subtypes of H5N1 and H5N6 in poultry-related environment of Hubei areas were investigated.Urban and rural live poultry markets,poultry farms,intensive livestock farms and other monitoring types of 103 counties in 17 cities were selected in Hubei.Wiping samples from cage surface,wiping samples from chopping board,fecal specimens and other environmental samples were collected and tested by real-time RT-PCR using primers and probes of influenza A,avian influenza of H5,N1 and N6 from December 2017 to March 2018.The avian influenza virus positive rate was compared among different monitoring sites,samples,time and regions.Totally,7132 environmental samples were collected in 1634 monitoring points with a positive rate of 2.24%.The positive rate of H5 avian influenza virus was the highest in urban and rural live poultry markets(3.44%,x^2=61.329,P<0.05)in 6 monitoring sites and wiping samples from chopping board(5.46%,x^2=67.072,P<0.05)in 6 sample types.H5N6 avian influenza viruses were detected more in eastern than western Hubei,and H5N6 avian influenza viruses were detected only in Xiangyang city of western Hubei.There were important high-risk places of human infection with H5 avian influenza virus in urban and rural live poultry markets and the poultry slaughtering plants.H5N6 has been the predominant subtype of H5 avian influenza viruses in the eastern and western Hubei and H5N6 avian influenza viruses were still present in a few areas of Hubei.Outbreaks of human H5N1 and H5N6 avian influenza remain at risk in Hubei province.

Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .

Selection Pressure on Haemagglutinin Genes of H9N2 Influenza Viruses from Different Hosts

Positive selection and differential selective pressure analyses were carried out to study Haemagglutinin (HA) genes of H9N2 influenza viruses from different hosts in this paper. Results showed that, although most positions in HAs were under neutral or purifying evolution, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection and some of them were even positively selected at the population level. In addition, there were always some positions differentially selected for viruses from different hosts. Both selection pressure working on HA codons and positions differentially selected might account for the extension of the host range and adaptations to different hosts of H9N2 influenza viruses.

Development of Neural Network for BLSOM Clustering of HA Genes of Avian Influenza Viruses Isolated in Guangdong Province

A neural network classification method,and a batch-learning self-organizing map(BLSOM),was established using trinucleotide and tetranucleotide in the hemagglutinin gene sequences of 25 avian influenza viruses isolated in Guangdong Province. Statistical analysis and normalization of the fragment number were done and MATLAB function was used to simulate the human brain thinking for self-organizing learning. When the number of training steps was 100 and above,the strains could be successfully clustered. H_1,H_3,H_5,H_7 and H_9 subtype strains fell within different classes,respectively,and the HA gene cluster map of H_3N_2 and H_7N_9strains was quite similar,suggesting that these strains shared the same origin; H_5N_1 strain was quite different in different years; H_1N_1 and H_9N_2 strains could be clustered into one group,indicating the natural recombinant variation in the two kinds of viruses,thereby providing a reference for high-risk strain screening and traceability.

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A(H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses(compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.

Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses,China,2021–2022

The recent concurrent emergence of H5N1,H5N6,and H5N8 avian influenza viruses(AIVs)has led to significant avian mortality globally.Since 2020,frequent human-animal interactions have been documented.To gain insight into the novel H5 subtype AIVs(i.e.,H5N1,H5N6 and H5N8),we collected 6102 samples from various regions of China between January 2021 and September 2022,and identified 41 H5Nx strains.Comparative analyses on the evolution and biological properties of these isolates were conducted.Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b,with 13 related to H5N1,19 to H5N6,and 9 to H5N8.Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8,exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015–2022 worldwide.H5N1 showed a higher rate of evolution in 2021–2022 and more sites under positive selection pressure in 2015–2022.The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations.Further hemagglutination inhibition assay suggested that some A(H5N1)viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains.Mammalian challenge assays demonstrated that the H5N8 virus(21GD001_H5N8)displayed the highest pathogenicity in mice,followed by the H5N1 virus(B1557_H5N1)and then the H5N6 virus(220086_H5N6),suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts.Based on the above results,we speculate that A(H5N1)viruses have a higher risk of emergence in the future.Collectively,these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b,contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Naturally occurring PA^(E206K)point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century.In this study,we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients.Sequence comparison of the three main protein subunits(PB2,PB1,and PA)of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution(E206K)was responsible for the observed impaired replication phenotype.Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo.Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature(39.5℃).Hence,this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Genetic and biological properties of H10Nx influenza viruses in China

H10 subtype avian influenza viruses(AIV)have been circulating in China for 40 years.H10 AIVs in China have expanded their host range from wild birds to domestic poultry and mammals,even human.Most of the H10 subtype AIVs reported in China were isolate from the southeast part.We isolated an H10N3 AIV,A/Chicken/Liaoning/SY1080/2021(SY1080),from live poultry market(LPM)in Liaoning Province of the Northeast China.SY1080replicated efficiently in mice lungs and nasal turbinates without prior adaptation.We systematically compared SY1080 with other H10 subtype isolates in China.Phylogenetic analysis showed that SY1080 and most of the H10strains belonged to the Eurasian lineage.H10 AIVs in China have formed 63 genotypes.SY1080 as well as the H10N3 strains from human infections belonged to G60 genotype.H10Nx AIV acquired multiple mammalian adaptive and virulence related mutations during circulation and the recent reassortants derived internal genes from chicken H9N2 AIVs.The H10Nx subtypes AIVs posed potential threat to public health.These results suggested we should strengthen the surveillance and evaluation of H10 subtype strains.

Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus

Recently,increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus(IAV)infections.Notably,some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans,posing a threat to public health.Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32(ANP32)is the most important one among them.However,role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.In the present study,a total of 10 feline ANP32(feANP32)splice variants(including 5 feANP32A,3 feANP32B,and 2 feANP32E)were obtained from domestic cats by RT-PCR.Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants,but all feANP32 proteins were primarily localized to cell nucleus.Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains,though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.In addition,the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.Collectively,our study systematically investigated the support activity of feANP32 on IAV,providing a clue for further exploring the mechanism of susceptibility of cats to IAV.

High-Value-Added Utilization of Turpentine:Screening of Anti-Influenza Virus Agents fromβ-Pinene Derivatives

Turpentine is a renewable and resourceful forest product.The deep processing and utilization of turpentine,particularly its primary componentβ-pinene,has garnered widespread attention.This study aimed to synthesize 40 derivatives ofβ-pinene,including nopinone,3-cyanopyridines of nopinone,myrtanyl acid,myrtanyl acylthioureas,and myrtanyl amides.We assessed the antiviral activities of theseβ-pinene derivatives against influenza virus A/Puerto Rico/8/34(H1N1)using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.Theβ-pinene derivatives were used before and after cellular infection with the influenza virus to evaluate their preventive and therapeutic effects against the H1N1 virus.The results showed that only compound 10o exhibited a preventive effect against the H1N1 virus with a half-maximal inhibitory concentration(IC50)value of 47.6μmol/L.Among the compounds,4e,4i,and 4l demonstrated therapeutic effects against cellular infection,with compound 4e displaying the most potent therapeutic effect(IC50=17.5μmol/L),comparable to the positive control ribavirin.These findings indicated that certainβ-pinene derivatives exhibited in vitro antiviral activity against the H1N1 influenza A virus,warranting further investigation as potential anti-influenza agents.

Pudilan Xiaoyan oral liquid regulates tissue inflammation and apoptosis in mice with influenza virus pneumonia

Background:The influenza A virus is the primary cause of respiratory infections and poses a global health risk.Pudilan Xiaoyan oral liquid(PDL)exhibits anti-inflammatory and immunomodulatory properties.PDL is commonly employed in clinical practice to manage upper respiratory tract infections.However,there is still much to uncover regarding its potential therapeutic mechanism.Methods:Institute of cancer research mice were infected with influenza A virus via nasal drip.The general state of the mice,lung index,and lung index inhibition rate were used to evaluate the efficacy of PDL.Enzyme-linked immunosorbent assay,western blotting,and immunohistochemistry were used to observe the presence of proteins and cytokines in the lung tissue.Apoptosis was evaluated using the TUNEL assay.Results:PDL improved the mental state of influenza A virus-infected mice,reduced the lung index,and inhibited viral replication.The expression of interleukin-1βand tumor necrosis factor-αwere decreased,whereas the expression of interleukin-10 in the lung tissue was increased due to PDL treatment.In addition,PDL treatment modulated Toll-like receptor 4 and MyD88 expressions in the lung tissues.PDL significantly reduced apoptosis and decreased cleaved caspase-3 and PARP levels,whereas increased B-cell lymphoma-2 expression in the lung tissue.Notably,the moderate-dose group of PDL exhibited a more pronounced effect.These findings indicate that PDL exerts a protective effect against pneumonia injury in influenza A virus-infected mice.Conclusion:PDL inhibited the inflammatory response and regulated apoptosis by regulating Toll-like receptor 4 and MyD88 protein expressions,thereby protecting the lung tissue from viral infection-induced lung tissue injury.