Identification and Analysis of Biomarkers Associated with Lipophagy and Therapeutic Agents for COVID-19.

BACKGROUND: Lipids, as a fundamental cell component, play an regulating role in controlling the different cellular biological processes involved in viral infections. A notable feature of coronavirus disease 2019 (COVID-19) is impaired lipid metabolism. The function of lipophagy-related genes in COVID-19 is unknown. The present study aimed to investigate biomarkers and drug targets associated with lipophagy and lipophagy-based therapeutic agents for COVID-19 through bioinformatics analysis. METHODS: Lipophagy-related biomarkers for COVID-19 were identified using machine learning algorithms such as random forest, Support Vector Machine-Recursive Feature Elimination, Generalized Linear Model, and Extreme Gradient Boosting in three COVID-19-associated GEO datasets: scRNA-seq (GSE145926) and bulk RNA-seq (GSE183533 and GSE190496). The cMAP database was searched for potential COVID-19 medications. RESULTS: The lipophagy pathway was downregulated, and the lipid droplet formation pathway was upregulated, resulting in impaired lipid metabolism. Seven lipophagy-related genes, including ACADVL, HYOU1, DAP, AUP1, PRXAB2, LSS, and PLIN2, were used as biomarkers and drug targets for COVID-19. Moreover, lipophagy may play a role in COVID-19 pathogenesis. As prospective drugs for treating COVID-19, seven potential downregulators (phenoxybenzamine, helveticoside, lanatoside C, geldanamycin, loperamide, pioglitazone, and trichostatin A) were discovered. These medication candidates showed remarkable binding energies against the seven biomarkers. CONCLUSIONS: The lipophagy-related genes ACADVL, HYOU1, DAP, AUP1, PRXAB2, LSS, and PLIN2 can be used as biomarkers and drug targets for COVID-19. Seven potential downregulators of these seven biomarkers may have therapeutic effects for treating COVID-19.

Silencing of V-ATPase-E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V-ATPase subunit E (V-ATP-E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V-ATP-E from D. citri had high homology with proteins from other insects. V-ATP-E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V-ATP-E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V-ATP-E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double-stranded RNA of D. citri V-ATP-E was safe to non-target parasitic wasps. These results suggest that V-ATP-E is an effective RNAi target that can be used in D. citri control to block CLas infection.

Analysis and identification of potential type II helper T cell (Th2)-Related key genes and therapeutic agents for COVID-19.

COVID-19 pandemic poses a severe threat to public health. However, so far, there are no effective drugs for COVID-19. Transcriptomic changes and key genes related to Th2 cells in COVID-19 have not been reported. These genes play an important role in host interactions with SARS-COV-2 and may be used as promising target. We analyzed five COVID-19-associated GEO datasets (GSE157103, GSE152641, GSE171110, GSE152418, and GSE179627) using the xCell algorithm and weighted gene co-expression network analysis (WGCNA). Results showed that 5 closely correlated modular genes to COVID-19 and Th2 cell enrichment levels, including purple, blue, pink, tan and turquoise, were intersected with differentially expressed genes (DEGs) and 648 shared genes were obtained. GO and KEGG pathway enrichment analyses revealed that they were enriched in cell proliferation, differentiation, and immune responses after virus infection. The most significantly enriched pathway involved the regulation of viral life cycle. Three key genes, namely CCNB1, BUB1, and UBE2C, may clarify the pathogenesis of COVID-19 associated with Th2 cells. 11 drug candidates were identified that could down-regulate three key genes using the cMAP database and demonstrated strong drugs binding energies aganist the three keygenes using molecular docking methods. BUB1, CCNB1 and UBE2C were identified key genes for COVID-19 and could be promising therapeutic targets.

Geraniin as a potential inhibitor of SARS-CoV-2 3CLpro.

Geraniin is a polyphenolic compound first isolated from Geranium thunbergii. The major protease (Mpro), namely 3 C-like protease (3CLpro), of coronaviruses is considered an attractive drug target as it is essential for the processing and maturation of viral polyproteins. Thus, our primary goal is to explore the efficiency of geraniin on 3CLpro of SARS-CoV-2 using the computational biology strategy. In this work, we studied the anti-coronavirus effect of geraniin in vitro and its potential inhibitory mode against the 3CLpro of SARS-CoV-2. We found that geraniin inhibited HCoV-OC43 coronavirus-infected cells during the attachment and penetration phases. Molecular docking and dynamics simulations exhibited that geraniin had a strong binding affinity and high stable binding to 3CLpro of SARS-CoV-2. Geraniin showed a strong inhibitory activity on coronavirus and may be a potential inhibitor of SARS-CoV-2 3CLpro.

Impact of Huanglongbing Pathogen Infection on the Amino Acid Composition in Both Citrus Plants and the Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) Diaphorina citri is the main vector of the pathogen Candidatus Liberibacter asiaticus (CLas), which is the causal agent of citrus Huanglongbing disease. Feeding by both ACP nymphs and adults on host plants allows them to obtain nutrition. Therefore, the nutritional content within the plant phloem is of much importance for the development and reproduction of ACP. The infection by pathogenic microbiomes may affect the amino acid contents of their host plants and then indirectly affect the biology of sap-feeding insects. In this study, we investigated the amino acid contents and their proportions in both CLas-infected and CLas-free citrus plants, ACP adults, and also in honeydew produced by ACP nymphs. Results showed that infection by CLas had a large impact on the amino acid species and proportion in all the tested target plants, ACP adults, and in the honeydew of ACP nymphs. The content of total amino acids in CLas-infected citrus was much higher than that of CLas-free citrus. However, CLas infection significantly reduced the proportion of essential amino acids (EAAs) in these plants. When feeding on CLas-infected citrus plants, ACP adults absorbed less total amino acids than those adults feeding on healthy plants, but the proportion of EAAs was significantly higher when they fed on CLas-infected citrus plants. The proportion of EAAs also significantly increased in the honeydew secreted by ACP nymphs that fed on CLas-infected citrus plants. However, EAA detection in the honeydew of ACP nymphs indicated that the utilization rate of EAAs by CLas positive ACP nymphs was reduced. Our study has revealed that CLas infection significantly affects the contents, proportion, and utilization efficiency of different amino acids in citrus plants, ACP adults, and nymphs, leading to a developmental pattern of ACP that is more conducive to CLas transmission.

Effect of paeoniflorin on acute lung injury induced by influenza A virus in mice. Evidences of its mechanism of action.

BACKGROUND: Influenza often leads to acute lung injury (ALI). Few therapeutics options such as vaccines and other antiviral drugs are available. Paeoniflorin is a monoterpene glucoside isolated from the roots of Paeonia lactiflora Pall. that has showed good anti-inflammatory and anti-fibrotic effects. However, it is not known whether paeoniflorin has an effect on influenza virus-induced ALI. PURPOSE: To investigative the protective effect and potential mechanism of paeoniflorin on ALI induced by influenza A virus (IAV). STUDY DESIGN AND METHODS: The anti-influenza activity of paeoniflorin in vitro was investigated. Influenza virus A/FM/1/47 was intranasally infected in mice to induce ALI, and paeoniflorin (50 and 100 mg/kg) was given orally to mice during 5 days, beginning 2 h after infection. On day 6 post-infection, body and lung weights, histology and survival were observed, and the lungs were examined for viral load, cytokine and cellular pathway protein expression. RESULTS: Results showed that paeoniflorin (50 and 100 mg/kg) reduced IAV-induced ALI. It reduces pulmonary oedema and improves histopathological changes in the lung, and also diminishes the accumulation of inflammatory cells in the lung. It was shown that paeoniflorin (50 and 100 mg/kg) alleviated IAV-induced ALI, as evidenced by improved survival in infected mice (40% and 50%, respectively), reduced viral titer in lung tissue, improved histological changes, and reduced lung inflammation. Paeoniflorin also improves pulmonary fibrosis by reducing the levels of pulmonary fibrotic markers (collagen type IV, alpha-smooth muscle actin, hyaluronic acid, laminin, and procollagen type III) and downregulating the expression levels of type I collagen (Col I) and type III collagen (Col III) in the lung tissues. Additionally, paeoniflorin inhibits the expression of αvß3, TGF-ß1, Smad2, NF-κB, and p38MAPK in the lung tissues. CONCLUSION: The results showed that paeoniflorin (50 and 100 mg/kg) protected against IAV-induced ALI, and the underlying mechanism may be related to the reduction of pro-inflammatory cytokine production and lung collagen deposition through down-regulation of activation of αvß3/TGF-ß1 pathway in lung tissue.

Puerarin reduces impairment of intestinal and adipose immune responses to influenza virus infection in mice.

Influenza is an acute viral respiratory disease that can also cause gastroenteritis-like symptoms, such as abdominal pain, nausea, vomiting, and diarrhea. Immune dysfunction of adipose tissue is involved in the occurrence and prognosis of influenza viral pneumonia. In this study, we analyzed intestinal and adipose immune responses in mice infected with influenza virus and found that the impairment of intestinal and adipose immunity to influenza virus infection could be reversed by treatment with puerarin, a medicinal compound isolated from Pueraria lobata (called "gegen" in Chinese). We found that the lungs, small intestines (duodenum, ileum, jejunum) and large intestines (colon and rectum) of infected mice showed obvious inflammatory lesions, with significantly increased levels of virus, inflammatory cytokines (interleukin [IL]-6, IL-17, and tumor necrosis factor-α), Toll-like receptors 3, 4, and 9, and integrin αvß3 and α4, and a decreased level of secreted IgA compared to the normal control group (NC) (P < 0.05-0.001). Influenza virus infected mesenteric lymph nodes and adipose tissue, and adipokines (leptin, visfatin, "chemerin", and adiponectin) of lung and mesenteric adipose tissue were dysregulated. Puerarin treatment reversed the impairment of the intestinal and adipose immune responses in mice infected with influenza virus. Our findings suggest that influenza virus can infect adipose tissue and lead to intestinal adipose immune dysfunction in normal-weight mice and that the impairment of the intestinal and adipose immune response to influenza virus infection can be reversed by puerarin treatment.

A potential antiviral activity of Esculentoside A against binding interactions of SARS-COV-2 spike protein and angiotensin converting enzyme 2 (ACE2).

The recent emergence of the novel coronavirus (SARS-CoV-2) has resulted in a devastating pandemic with global concern. However, to date, there are no regimens to prevent and treat SARS-CoV-2 virus. There is an urgent need to identify novel leads with anti-viral properties that impede viral pathogenesis in the host system. Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, is known to exhibit diverse pharmacological properties, especially anti-inflammatory activity. To our knowledge, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) to enter host cells. This is mediated through the proteins of SARS-CoV-2, especially the spike glycoprotein receptor binding domain. Thus, our primary goal is to prevent virus replication and binding to the host, which allows us to explore the efficiency of EsA on key surface drug target proteins using the computational biology paradigm approach. Here, the anti-coronavirus activity of EsA in vitro and its potential mode of inhibitory action on the S-protein of SARS-CoV-2 were investigated. We found that EsA inhibited the HCoV-OC43 coronavirus during the attachment and penetration stage. Molecular docking results showed that EsA had a strong binding affinity with the spike glycoprotein from SARS-CoV-2. The results of the molecular dynamics simulation revealed that EsA had higher stable binding with the spike protein. These results demonstrated that Esculentoside A can act as a spike protein blocker to inhibit SARS-CoV-2. Considering the poor bioavailability and low toxicity of EsA, it is suitable as novel lead for the inhibitor against binding interactions of SARS-CoV-2 of S-protein and ACE2.

Antiviral activity of puerarin as potent inhibitor of influenza virus neuraminidase.

Puerarin is a major isofiavone compound isolated from the root of Pueraria lobata. It was reported that puerarin had antioxidant, antiinflammatory, antitumor, cholesterol lowering, liver protective, and neuroprotective properties. However, few studies have explored the antiviral effect of puerarin and its target mechanism related to influenza virus. Here, the antiinfluenza activity of puerarin in vitro and in vivo and its mode of action on the potential inhibition of neuraminidase (NA) were investigated. Puerarin displayed an inhibitory effect on A/FM/1/1947(H1N1) (EC50 = 52.06 µM). An indirect immunofluorescence assay indicated that puerarin blocked the nuclear export of viral NP. The inhibition of NA activity confirmed that puerarin can block the release of newly formed virus particles from infected cells. Puerarin (100 and 200 mg/kg/d) exhibited effective antiviral activity in mice, conferring 50% and 70% protection from death against H1N1, reducing virus titers, and effectively alleviating inflammation in the lungs. The molecular docking results showed that puerarin had a strong binding affinity with NA from H1N1. The results of the molecular dynamics simulation revealed that puerarin had higher stable binding at the 150-loop region of the NA protein. These results demonstrated that puerarin acts as a NA blocker to inhibit influenza A virus both in cellular and animal models. Thus, puerarin has potential utility for the treatment of the influenza virus infection.

3D-quantitative structure-activity relationship and antiviral effects of curcumin derivatives as potent inhibitors of influenza H1N1 neuraminidase.

Curcumin derivatives have been shown to inhibit replication of human influenza A viruses (IAVs). However, it is not clear whether curcumin and its derivatives can inhibit neuraminidase (NA) of influenza virus. In this study, a meaningful 3D quantitative structure-activity relationship model (comparative molecular field analysis R2 = 0.997, q2 = 0.527, s = 0.064, F = 282.663) was built to understand the chemical-biological interactions between their activities and neuraminidase. Molecular docking was used to predict binding models between curcumin derivatives and neuraminidase. Real-time polymerase chain reactions showed that the five active curcumin derivatives might have direct effects on viral particle infectivity in H1N1-infected lung epithelial (MDCK) cells. Neuraminidase activation assay showed that five active curcumin derivatives decreased H1N1-induced neuraminidase activation in MDCK cells. Indirect immunofluorescence assay indicated that two active curcumin derivatives (tetramethylcurcumin and curcumin) down-regulated the nucleoprotein expression. Curcumin inhibited IAV in vivo. The therapeutic mechanism of curcumin in the treatment of influenza viral pneumonia is related to improving the immune function of infected mice and regulating secretion of tumor necrosis-α, interleukin-6, and interferon-γ. These results indicate that curcumin derivatives inhibit IAV by blocking neuraminidase in the cellular model and curcumin also has anti-IAV activity in the animal model.

Polygalasaponin F treats mice with pneumonia induced by influenza virus.

BACKGROUND: Influenza is an acute viral respiratory illness that causes high morbidity and mortality globally. Therapeutic actions are limited to vaccines and a few anti-viral drugs. Polygala (P.) japonica herba is rich in Polygalasaponin F (PSF, C53H86O23), used for acute bronchitis, pharyngitis, pneumonia, amygdalitis, and respiratory tract infections treatment in China. Hypercytokinemia is often correlated with severe pneumonia caused by several influenza viruses. PSF was reported to have anti-inflammatory effects and its mechanism is associated with the nuclear factor (NF)-κB signaling pathway. The action of PSF to alleviate pulmonary inflammation caused by influenza A virus (IAV) infection requires careful assessment. In the present study, we evaluated the effect and mechanism of PSF on mice with pneumonia caused by influenza H1N1 (A/FM/1/47). METHODS: Mice were infected intranasally with fifteen 50% mouse lethal challenge doses (MLD50) of influenza virus. BALB/c mice were treated with PSF or oseltamivir (oral administration) for 2 h post-infection and received concomitant treatment for 5 days after infection. On day 6 post-infection, 10 mice per group were killed to collect related samples, measure body weight and lung wet weight, and detect the viral load, cytokine, prostaglandins, pathological changes, and cell pathway protein expression in the lungs. In addition, the survival experiments were carried out to investigate the survival of mice. The expression profile of cell pathway proteins was detected and analyzed using a broad pathway antibody array and confirmed the findings from the array by western blotting. RESULTS: Polygalasaponin F and oseltamivir can protect against influenza viral infection in mice. PSF and oseltamivir significantly relieved the signs and symptoms, reduced body weight loss, and improved the survival rate of H1N1-infected mice. Moreover, PSF efficiently decreased the level of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-4, interferon (IFN)-γ, thromboxane A2 (TXA2), and prostaglandin E2 (PGE2) in lung tissues of mice infected with influenza virus (p < 0.05-0.01). Oseltamivir had a similar effect to lung cytokine of PSF, but did not decrease the levels of TXA2 and PGE2. There was a twofold or greater increase in four cell pathway protein, namely NF-κB p65 (2.68-fold), I-kappa-B-alpha (IκBα) (2.56-fold), and MAPK/ERK kinase 1 (MEK1) (7.15-fold) assessed in the array induced by influenza virus. Western blotting showed that the expression of these proteins was significantly decreased in lung after influenza virus challenge in PSF and oseltamivir-treated mice (p < 0.05-0.01). CONCLUSION: Polygalasaponin F appears to be able to augment protection against IAV infection in mice via attenuation of pulmonary inflammatory responses. Its effect on IAV-induced pulmonary inflammation was associated with suppression of Raf/MEK/ERK and NF-κB expressions.

Intranasal co-administration of 1,8-cineole with influenza vaccine provide cross-protection against influenza virus infection.

BACKGROUND: Vaccination is the most efficient means for protection against influenza. However, the various vaccines have low efficacy to protect against pandemic strains because of antigenic drift and recombination of influenza virus. Adjuvant therapy is one of the attempts to improve influenza vaccine effective cross-protection against influenza virus infection. Our previous study confirmed that 1,8-cineole inhibits the NF-κB, reduces pro-inflammatory cytokines, and relieves the pathological changes of viral pneumonia in mice infected with influenza virus. HYPOTHESIS/PURPOSE: 1,8-cineole, administered via intranasal (i.n.) route, may also have the capacity to be an adjuvant of the influenza vaccine. This study was designed to investigate the potential use of i.n. co-administration of 1,8-cineole, a major component of the Eucalyptus essential oils, with influenza vaccine and whether could provide cross-protection against influenza virus infection in a mouse model. STUDY DESIGN: I.n. co-administration of 1,8-cineole in two doses (6.25 and 12.5 mg/kg) with influenza vaccine was investigated in a mouse model in order to see whether it could provide cross-protection against influenza virus infection. METHODS: The mice were intranasally immunized three times at the 0, 7 and 14 day with vaccine containing 0.2 µg hemagglutinin (HA) and/or without 1,8-cineole. Seven days after the 3rd immunization dose, the mice were infected with 50 µl of 15 LD50 (50% mouse lethal dose) influenza virus A/FM/1/47 (H1N1). On day 6 post-infection, 10 mice per group were sacrificed to collect samples, to take the body weight and lung, and detect the viral load, pathological changes in the lungs and antibody, etc. The collected samples included blood serum and nasal lavage fluids. In addition, the survival experiments were carried out  to investigate the survival of mice. RESULTS: Mice i.n. inoculated with influenza vaccine and 12.5 mg/kg 1,8-cineole increased the production of influenza-specific serum immunoglobulin (Ig) G2a antibodies, stimulated mucosal secretive IgA (s-IgA) responses at the nasal cavity, improved the expression of respiratory tract intraepithelial lymphocytes (IELs) in the upper respiratory tract, and promoted dendritic cell (DC) maturation and the expression of co-stimulatory molecules cluster of differentiation (CD)40, CD80 and CD86 in peripheral blood. Importantly, mice that had received 1,8-cineole-supplemented influenza vaccine showed longer survival time, milder inflammation, less weight loss and mortality rate and lower lung index and viral titers compared to that of mice immunized a non-1,8-cineole-adjuvanted split vaccine. Thus, i.n. immunization with 1,8-cineole-adjuvanted vaccine induces a superior cross-protective immunity against infection with influenza than an inactivated vaccine only. CONCLUSION: These results suggest that 1,8-cineole (12.5 mg/kg) has a cross-protection against influenza virus, co-administered with inactivated influenza viral antigen in a mouse model.

Ribavirin attenuates the respiratory immune responses to influenza viral infection in mice.

Ribavirin is a broad-spectrum antiviral agent that is used against RNA and DNA viruses and has been reported to inhibit infection by influenza A and B virus in vitro and in vivo. Studies have shown that ribavirin can lower convalescent antibody titers in young children hospitalized with influenza. Here, we report that ribavirin administration in juvenile mice significantly attenuated respiratory immune responses, production of total IgA and hemagglutinin (HA)-specific secretory IgA responses on the mucosal surface. In contrast, systemic IgG and IgA responses were not affected. Ribavirin significantly suppressed toll-like receptor 2 and 4 expression in the lung and decreased the level of IL-1ß, IL-6, TNF-α, and IFN-γ in lung tissues of mice infected with influenza virus. Our findings suggest ribavirin appears to be able to inhibit viral replication and, as a result, TLR and cytokine expression are not up-regulated, attenuating inflammation as well as the respiratory tract's immune response.

Combinations of 1,8-cineol and oseltamivir for the treatment of influenza virus A (H3N2) infection in mice.

It is need for development of new means against influenza virus due to the lack of efficacy of available therapeutic strategies. In previous research, 1,8-cineol exert its inhibition of nuclear factor (NF)-κB, the main regulator of cytokine and chemokine production in influenza, and anti-inflammatory activity. These fact supports and helps establish the hypothesis that 1,8-cineol may have synergism with an antiviral on influenza virus infection. The combined effect of 1,8-cineol with oseltamivir in a mouse type A influenza virus (Victoria/3/75,H3N2) model were examined. We initially tested combinations of 1,8-cineol (30, 60, and 120 mg/kg/day) and oseltamivir (0.1, 0.2, and 0.4 mg/kg/day). In addition, the 0.4 mg/kg/day of oseltamivir combined with 120 mg/kg of 1,8-cineol was selected for further combination studies. Oseltamivir was 30%, 40%, and 60% protective at 0.1, 0.2, and 0.4 mg/kg/d. Combinations of 1,8-cineol (30, 60, and 120 mg/kg/d) and oseltamivir (0.1, 0.2, and 0.4 mg/kg/d) increased the number of survivors and mean survival time (MST) following combination treatment was greater than monotherapy alone. Three dimensional analysis of drug interactions using the MacSynergy method showed a strong synergistic effect of these drug combinations. Survival, MST, lung parameters (lung index, viral titers, and pathology), and cytokines (IL-10, TNF-α, IL-1ß, and IFN-γ) expression in lung demonstrated the high effectiveness of the combination. Combined treatment was associated with longer MST and more reduced cytokine levels than oseltamivir alone. These data demonstrate that combinations of 1,8-cineol and oseltamivir have synergistic effect against influenza A virus (H3N2) infection.

1, 8-Cineol Protect Against Influenza-Virus-Induced Pneumonia in Mice.

1,8-Cineol is a major monoterpene principally from eucalyptus essential oils and has been shown to exert anti-inflammatory, antiviral, and inhibitory of nuclear factor (NF)-kB effect. In the present study, we evaluated the effect of 1,8-cineol on mice infected with influenza A virus. We found that 1,8-cineol protects against influenza viral infection in mice. Moreover, 1,8-cineol efficiently decreased the level of IL-4, IL-5, IL-10, and MCP-1 in nasal lavage fluids and the level of IL-1ß, IL-6, TNF-α, and IFN-γ in lung tissues of mice infected with influenza virus. The results also showed that 1,8-cineol reduced the expression of NF-kB p65, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 in lung tissues. Thus, 1,8-cineol appears to be able to augment protection against IFV infection in mice via attenuation of pulmonary inflammatory responses.

Oral administration of patchouli alcohol isolated from Pogostemonis Herba augments protection against influenza viral infection in mice.

Seasonal influenza A infection results in considerable morbidity and mortality. The limited efficacy of available therapeutic strategies stresses the need for development and study of new molecules against influenza virus (IFV). Patchouli alcohol (PA), the major chemical constituent of Pogostemonis Herba, was previously found to strongly inhibit influenza H1N1 replication in vitro. In the present study, the in vivo anti-IFV effect of PA was investigated. In a mouse model infected with lethal levels of FM1, oral administration of PA (20 mg/kg to 80 mg/kg) for 7 d post IFV infection significantly increased the survival rate and survival time. For IFV infection at nonlethal levels, the quantity of IFV in the lungs 5 d after infection was significantly reduced after PA (20 mg/kg to 80 mg/kg) administration. Anti-IFV IgA, IgM, and IgG titers in serum on day 6 were significantly higher in the PA-treated group than the IFV-control group. Anti-IFV immune response augmentation was further confirmed by the elevated production of CD3+, CD4+, and CD8+ T cell levels in blood. Furthermore, the levels of inflammatory cytokines, including TNF-alpha, IL-10 and IFN-gamma in serum of mice, were regulated. Lung inflammation was reduced significantly after PA administration, and the effect may be mediated, at least in part, by regulating the lung levels of inflammatory cytokines. Thus, oral administration of PA appears to be able to augment protection against IFV infection in mice via enhancement of host immune responses, and attenuation of systemic and pulmonary inflammatory responses.