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.

A_3B亚型检测与血清学分析

1资料与方法患者,女,53岁,左乳腺癌术后9年,2014年11月因颈椎转移入院治疗。患者血型鉴定为AB型,RhD、RhE阳性。卡式法检测发现其红细胞与抗-A出现＂混合视野＂凝集,需进一步结合血清学吸收放散试验等进行鉴定。ABODE血型检测卡（微柱凝胶）,ABO血型反定型试剂盒（人血红细胞）,由长春博迅生物技术有限责任公司提供;抗A、抗B血型定型试剂（单克隆抗体）,A2B型红细胞,

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice

Objective Interferon-induced transmembrane protein 3(IFITM3)is an important member of the IFITM family.However,the molecular mechanisms underlying its antiviral action have not been completely elucidated.Recent studies on IFITM3,particularly those focused on innate antiviral defense mechanisms,have shown that IFITM3 affects the body’s adaptive immune response.The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo.Methods We performed proteomics,flow cytometry,and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer(NK)cell numbers,their activation,and their immune function in the lungs of Ifitm3-/-and wild-type mice.Results Ifitm3-/-mice developed more severe inflammation and apoptotic responses compared to wild-type mice.Moreover,the NK cell activation was higher in the lungs of Ifitm3-/-mice during acute influenza infection.Conclusions Based on our results,we speculate that the NK cells are more readily activated in the absence of IFITM3,increasing mortality in Ifitm3-/-mice.

Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China

One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

A Novel Reassortant H3N8 Influenza Virus Isolated from Drinking Water for Duck in a Domestic Duck Farm in Poyang Lake Area

Objective To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011. Methods The virus was cultivated by specific pathogen free （SPF） chicken embryo eggs and was subtyped into hemagglutinin （HA） and neuraminidase （NA） by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted. Results The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB2 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr225Ala of the M1 protein implied the potential of pathogenicity increase in mice. Conclusion The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.

A Novel Reassortant H2N3 Influenza Virus Isolated from China

Objective To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market （LPM） in 2009 in Guangdong province of China. Methods PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed. Results The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus. Conclusion market. Its A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry potential impacts and evolution should be closely monitored.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Clinical Predictors for Diagnosing Pandemic (H1N1) 2009 and Seasonal Influenza (H3N2) in Fever Clinics in Beijing,China

Objective Symptomatic predictors of influenza could assess risks and improve decisions about isolation and outpatient treatment. To develop such predictors, we undertook a prospective analysis of pandemic （HIN1） 2009 and seasonal influenza （H3N2） in patients attending fever clinics. Methods From 1 May 2009 to 1 January 2010, all adult patients admitted to fever clinics for suspected influenza, confirmed by real time RT-PCR, were enrolled. Predictors of influenza virus infection were selected with logistic regression models. Measures of sensitivity, specificity, positive and negative likelihood ratios （LRs） were calculated to identify the best predictors. Results The clinical features and routine blood test results of influenza （H1N1） 2009 and seasonal influenza were similar. The positive and negative LRs of current US CDC influenza-like illness （ILl） criteria were modest in predicting influenza infection. Our modified clinic predictors improved the ability of the positive and negative LRs to recognize pandemic （HIN1） 2009 and seasonal influenza. The revised criteria are： fever ~ 38 ~C accompanied by at least one of the following--cough, arthralgia or relative iymphopenia. Conclusion Patients with symptoms and signs that meet the new criteria are likely to have influenza and timely antiviral therapy may be appropriate. In addition, physicians should ascertain if influenza is circulating within the community or if there is a contact history of influenza and combine this information with the newly developed criteria to clinically diagnose influenza.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine

Objective To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. Methods Mice were immunized intranasally with PBS(negative control), or H3N2 split vaccine(3 μg/mouse) alone, or CTA1-DD(5 μg/mouse) alone, or H3N2 split vaccine(3 μg/mouse) plus CTA1-DD(5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine(3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. Results H3N2 split vaccine formulated with CTA1-DD could elicit higher Ig M, Ig G and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory Ig A titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. Conclusion CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Synthesis and Anti-influenza Virus Activity of Ethyl 6-Bromo-5-hydroxyindole-3-carboxylate Derivatives

A series of ethyl 6-bromo-5-hydroxyindole-3-carboxylate derivatives were synthesized and their in vitro anti-influenza virus activity was evaluated. All the compounds were characterized by 1H NMR and MS.

甘露消毒丹调控PI3K/Akt信号通路干预甲型流感病毒感染的作用机制

目的 基于网络药理学探讨甘露消毒丹治疗甲型流感病毒感染的活性成分及作用机制,并用动物实验加以验证。方法 利用中药系统药理学数据库与分析平台(TCMSP)获取和筛选药物有效成分,利用TCMSP及Swiss Target Prediction数据库预测潜在作用靶点。利用比较毒物基因组学数据库(CTD)、疾病相关基因与突变位点数据库(DisGeNET)、GeneCards数据库、在线人类孟德尔遗传数据库(OMIM)获得甲型流感病毒感染的疾病靶点,将药物预测潜在作用靶点与疾病靶点取交集获得交集靶点。利用Cytoscape3.9. 1构建药物-成分-靶点网络并进行拓扑属性分析,预测中药主要活性成分。利用蛋白互作数据库(STRING)及Cytoscape3.9. 1绘制蛋白质-蛋白质相互作用(PPI)网络图,获取关键作用靶点。利用Metascape数据库进行基因本体(GO)功能以及京都基因和基因组百科全书(KEGG)通路富集分析。将C57BL/6J小鼠分为空白组、模型组、中药组及对照组,每组5只,甲型流感病毒PR8滴鼻感染小鼠构建感染模型,中药组予以甘露消毒丹灌胃,对照组予磷酸奥司他韦灌胃。观察肺指数、肺组织病理变化,实时荧光定量逆转录PCR (RTqPCR)法检测肺组织磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B (AKT)基因表达,双抗体夹心酶联免疫吸附法(ELISA)检测肺组织IL-6、IL-1β、TNF-α。结果 共筛选出甘露消毒丹125个化合物作为候选有效成分,获得286个潜在作用靶点,2 223个甲型流感病毒相关疾病靶点,61个交集靶点。药物-成分-靶点网络得到槲皮素、山奈酚、汉黄芩素、木犀草素、黄芩素、葛花苷元、金合欢素、异鼠李素、去氢二异丁香酚、柚皮素等10个关键成分。PPI网络得到TNF、IL-6、IL-1β、AKT1、TP53、JUN、HIF1A、PTGS2、MMP9、ESR1等10个关键靶点。GO功能富集分析共获得条目54条,其中生物过程20条,细胞组分14条,分子功能20条;KEGG通路富集分析共获得条目共20条,其中包括AGE-RAGE信号通路、癌症通路、松弛素信号通路、PI3K-Akt信号通路等。与空白组比较,模型组肺指数高(P<0.05);与模型组比较,中药组、对照组肺指数低(P<0.05)。模型组小鼠肺组织间质水肿,大量淋巴细胞、单核细胞浸润,肺泡壁明显增宽,肺泡融合。中药组及对照组小鼠肺组织间质水肿减轻,淋巴细胞、单核细胞浸润减少,肺泡壁增宽减轻,部分肺泡融合。与空白组比较,模型组小鼠肺组织中PI3K、AKT mRNA表达高(P<0.05)。与模型组比较,中药组PI3K、AKT mRNA表达低(P<0.05),对照组PI3K mRNA表达低(P<0.05)。与空白组比较,模型组肺组织中IL-6、IL-1β、TNF-α水平高(P<0.05)。与模型组比较,中药组肺组织中IL-6、IL-1β、TNF-α水平低(P<0.05);对照组肺组织中IL-6、IL-1β水平低(P<0.05)。结论 甘露消毒丹可通过抑制甲型流感病毒感染小鼠炎症反应改善肺损伤,其作用机制可能与抑制PI3K/Akt信号通路的过度激活有关。

犬流感病毒H_(3)N_(2)和H_(3)N_(8)亚型流行病学研究进展

自2004年首次在美国发现犬流感病毒(canine influenza virus,CIV)以来,经过不断的演变进化和跨物种的传播,CIV已在全球范围内广泛传播。由于犬对多种流感病毒易感,流感病毒对犬的影响及其产生人畜共患的大流行性流感的风险不容忽视。文章就CIV的病原学及较稳定传播的两种亚型CIV(H_(3)N_(2)和H_(3)N_(8))的流行病学进行综述,旨在为CIV的预防、诊断以及疫苗研发等提供参考。

羚珠散抗感冒病毒诱发感染的体内外活性及作用机制

目的研究羚珠散抗感冒病毒诱发感染的体内外活性及作用机制。方法采用CCK8法检测不同浓度羚珠散对流感病毒A/FM/1/47(H1N1)、A/Beijing/32/92(H3N2)、奥司他韦耐药甲型流感病毒株(H1N1-OC)及人呼吸道合胞病毒(RSV)感染的HEP-2和MDCK细胞的保护作用。此外,采用流感病毒A/FM/1/47(H1N1)感染小鼠,探究羚珠散治疗对小鼠存活率、肺指数、肺损伤和肺组织病毒滴度的影响,通过分析小鼠血清及肺组织炎症因子IFN-γ、IL-1β和TNF-α的含量及肺组织NLRP-3/caspase-1炎症小体通路的活化情况,探讨羚珠散抗流感病毒感染的作用机制。结果体外实验中,羚珠散对4株受试病毒株均表现出良好的抑制效果,尤其对H1N1-OC和RSV的抑制效果较为明显;同时,羚珠散能够降低感染小鼠肺组织的病毒载量,抑制NLRP-3/caspase-1炎症小体通路的活化,从而减轻病毒感染造成的小鼠死亡及肺组织损伤。结论羚珠散对感冒病毒诱发的感染具有良好的抑制效果。

槲皮素对甲型H1N1流感病毒诱导的A549细胞凋亡效应酶Caspase-3的影响

目的观察银杏叶主要活性成分槲皮素对甲型H1N1流感病毒感染的人肺上皮瘤细胞A549凋亡效应酶Caspase-3的影响。方法采用MTT法测定H1N1毒力、槲皮素细胞毒性作用、槲皮素对H1N1致A549细胞病变的抑制作用。然后用100 TCID50甲型H1N1感染A549 2 h后换用含10 mg/L槲皮素维持液继续培养,于感染后4、12、24、48 h收集细胞,提取细胞总蛋白,采用Western blot方法,应用Image-Pro Plus测定Caspase-3与β-actin灰度值,两者灰度比值为Caspase-3的相对表达量。结果甲型H1N1 A549的TCID50为10-4.75,槲皮素A549的最大无毒浓度为40 mg/L,槲皮素抑制甲型H1N1致A549细胞病变的最小有效浓度为10 mg/L。在甲型H1N1感染A549细胞后4～48 h内,槲皮素可显著抑制Caspase-3蛋白表达,具有明显的抗凋亡作用。结论槲皮素可通过抑制Caspase-3含量或活性而发挥抗病毒感染作用。

黄芩苷对FM1肺炎小鼠肺组织细胞凋亡FAS/FAS-L系统的影响

目的研究黄芩苷对FM1肺炎小鼠肺组织细胞凋亡FAS/FAS-L系统的影响,探索黄芩苷抗流感病毒感染机制。方法制备小鼠甲型流感病毒性肺炎模型,通过死亡保护实验确定黄芩苷的最小有效浓度,TUNEL法观察小鼠肺组织细胞凋亡情况,RT-PCR技术检测肺组织FAS(CD95)、FAS-L(CD178)、Caspase-3 mRNA表达,Western blot方法测定肺组织FAS、FAS-L、Caspase-3蛋白表达。结果与模型组相比,黄芩苷187.5、375 mg.kg-1剂量均能明显降低肺组织细胞凋亡率,明显降低肺组织FAS、FAS-L及Caspase-3 mRNA和蛋白的表达(P<0.05)。结论黄芩苷能够明显抑制FM1肺炎小鼠肺组织细胞的凋亡,其可能通过影响细胞凋亡受体途径FAS/FAS-L系统而发挥抗流感病毒感染作用。

几种化学药物对银杏种子发芽力的影响

通过不同浓度的 Ca Cl2 、KNO3、GA3溶液对银杏 Ginkgo biloba L.种子浸种 ,研究其种子的发芽力。结果表明 :Ca Cl2 、KNO3、GA3在不同程度上提高银杏种子的发芽率和发芽势。三者浸种的最适浓度分别为 2 5 mm ol/ L、0 .4mol/ L 和 5 0 0 m g/ L。其中以 GA3的处理效果最好 ,用 5 0 0 mg/ L 浓度处理时可使银杏种子发芽率和发芽势分别提高 33.3%、2 4.9% ,而且幼苗生长健壮。同时用这 3种化学药物浸种还能降低烂种率、提早发芽。

金刚烷胺、病毒唑和鱼腥草三者联用时的协同抗流感病毒作用

The research of the cooperative antivirus activities was carried out using amantadine, ribavirin and herby houttuynia. The inhibitory effect of CPE caused by influenza virus A\-3 in MDCK cell and the therapeutic effect on BALB/c mice pneumonia caused by influenza virus FM1 were examined and analyzed when amantadine, ribavirin and herby houttuynia were used alone or associatively. The results show that the concentrations of amantadine,ribavirin and herby houttuynia with same effect were 256, 512 and 1024 times cooperatively as many as alone in vitro, respectively.The ablities of treating pneumonia of mice when three kinds of drugs were used cooperatively were far higher than those alone in BALB/c mice.

疏风宣肺方和解表清里方干预流感病毒性肺炎小鼠细胞凋亡的研究

目的病毒感染宿主细胞后,细胞凋亡天门冬氨酸特异性半胱氨酸蛋白(caspase)途径激活限制病毒复制,但甲型流感病毒感染引起的细胞凋亡的分子机制尚不完全清楚。探讨流感病毒性肺炎小鼠肺组织细胞凋亡相关的基因及2种不同抗流感病毒方药的治疗作用。方法制备甲型流感病毒性肺炎小鼠模型,随机分为空白组、肺炎模型组、奥司他韦对照组、疏风宣肺方高、中、低剂量组,解表清里方高、中、低剂量组。提取各组小鼠肺组织总RNA,采用基因组芯片筛选出与凋亡密切相关的差异表达基因,并用荧光定量PCR对天门冬氨酸特异性半胱氨酸蛋白基因进行验证。结果肺炎模型组肺组织中差异表达基因casp-3、casp-8、casp-9、Fas、FasL、TNF、TNFrasf1a、Tradd、IL1a、IL1b、IL1r1、IL1r2、casp-7明显上调,与空白组比较差异有统计学意义。疏风宣肺方中剂量组差异表达基因casp-8、casp-7、Fas、FasL、TNF、TNFrasf1a、Tradd、IL1a、IL1b、IL1r1、IL1r2明显下调。疏风宣肺方低剂量组下调上述基因的作用强于解表清里方低剂量组,疏风宣肺方中剂量组治疗效果优于解表清里方中剂量组。疏风宣肺方低剂量组与解表清里方中剂量组对casp-3、-8、-9表达有显著抑制,差异有统计学意义(P<0.01)。不同剂量疏风宣肺方的治疗效果与解表清里方比较如下:疏风宣肺方低剂量组>疏风宣肺方中剂量组>解表清里方中剂量组>解表清里方低剂量组。结论疏风宣肺方对细胞凋亡调控基因的调控作用强于解表清里方,上调casp-3、-8、-9表达,对抗流感病毒感染后的细胞凋亡较强,对甲型流感病毒性肺炎小鼠治疗作用优于解表清里方。

菊蓝感冒冲剂抗病毒作用实验研究

目的：研究菊蓝感冒冲剂对流感病毒甲型、乙型和腺病毒３ 型的抑制作用。方法：用鸡胚孵育法，进行菊蓝感冒冲剂对流感甲型、乙型病毒的直接杀伤及抑制病毒繁殖作用，用传代Ｈｅｌａ 细胞的细胞培养法，研究菊蓝感冒冲剂对腺病毒３型的直接杀伤及抑制病毒繁殖作用。结果：菊蓝感冒冲剂对流感甲型、乙型病毒的直接杀伤及抑制病毒繁殖均有明显作用（ Ｐ＜０．００１），对腺病毒３ 型的繁殖有抑制作用，而对腺病毒３ 型的直接杀伤作用无效。结论：菊蓝感冒冲剂具有一定的体外抗流感甲型、乙型病毒和腺病毒３ 型的作用。