Anti-Influenza Virus Activity of Myrica rubra Leaf Ethanol Extract Evaluated Using Madino-Darby Canine Kidney (MDCK) Cells

Myrica rubra leaf ethanol extract was added to culture medium of Madino-Darby canine kidney (MDCK) cells inoculated with influenza virus, and the inhibition of influenza virus replication was measured. Myrica rubra leaf ethanol extract showed anti-influenza virus activity irrespective of the hemagglutinin antigen type in the influenza virus type A (H1N1), its subtype (H3N2), and type B.     

Antiviral effect of flavonol glycosides isolated from the leaf of Zanthoxylum piperitum on influenza virus

The ethanol extract of Zanthoxylum piperitum (L.) DC. showed in vitro antiviral activity against influenza A virus. Three flavonol glycosides were isolated from the EtOAc fraction of Z. piperitum leaf by means of activity-guided chromatographic separation. Structures of isolated compounds were identified as quercetin 3-O-β-D-galactopyranoside (1), quercetin 3-O-α-L-rhamnopyranoside (2), kaempferol 3-O-α-L-rhamnopyranoside (3) by comparing their spectral data with literature values. The anti-influenza viral activity of isolates was evaluated using a plaque reduction assay against influenza A/NWS/33 (H1N1) virus. The compounds also were subjected to neuraminidase inhibition assay in influenza A/NWS/33 virus. Compounds 1–3 exhibited antiviral activity against an influenza A virus in vitro, and inhibited the neuraminidase activity at relatively high concentrations.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

杨梅树皮黄酮的提取及体外抗氧化研究

采用乙醇热回流提取法对杨梅树皮中总黄酮进行提取,通过正交试验确定了杨梅树皮总黄酮乙醇热回流提取法的最佳工艺条件:以28目的杨梅树皮粉为原料,料液比为1∶60,用60%的乙醇溶液在60℃的水浴中提取2.5h,提取率达6.86%。利用清除DPPH自由基的能力和还原力作为指标测定了杨梅树皮总黄酮的抗氧化活性。结果表明,杨梅树皮黄酮有较强的抗氧化能力。     

Xanthones from Polygala karensium inhibit neuraminidases from influenza A viruses

The emergence of the H1N1 swine flu pandemic has the possibility to develop the occurrence of disaster- or drug-resistant viruses by additional reassortments in novel influenza A virus. In the course of an anti-influenza screening program for natural products, 10 xanthone derivatives (1-10) were isolated by bioassay-guided fractionation from the EtOAc-soluble extract of Polygala karensium. Compounds 1, 3, 5, 7, and 9 with a hydroxy group at C-1 showed strong inhibitory effects on neuraminidases from various influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, these compounds reduced the cytopathic effect of H1N1 swine influenza virus in MDCK cells. Our results suggest that xanthones from P. karensium may be useful in the prevention and treatment of disease by influenza viruses.     

杨梅根系和土壤微生物量碳、氮、磷生态化学计量随林龄的变化

利用空间代替时间的方法,研究了浙江省仙居县3、9、14、21年生根系和土壤微生物量碳(C)、氮(N)、磷(P含量及化学计量比。结果表明,杨梅根系C、C:N在不同林龄间没有显著差异;随着林龄的增加,根系N、P含量降低,而C:P、N:P则增大; 21年生杨梅根系N含量显著低于3年生(P<0.05),磷含量显著低于其他林龄(P<0.05); 21年生杨梅根系C:P、N:P显著高于其他林龄(P<0.05)。从根系N:P的变化可知,随着林龄增大,杨梅生长受到P的限制更加明显。土壤微生物量碳(MBC)、微生物量氮(MBN)、微生物量磷(MBP)含量和MBC:MBN、MBC:MBP表现为先明显下降而后略有升高,而MBN:MBP总体呈现上升趋势。3年生杨梅MBC含量和MBC:MBN显著高于其他林龄(P<0.05),MBN、MBP含量显著高于9年生(P<0.05); 9年生MBC:MBP显著低于其他林龄(P<0.05); 3年生0—10 cm土层的MBN:MBP显著低于其他林龄(P<0.05),而21年生的10—30 cm土层的MBN:MBP显著高于其他林龄(P...     

Cryptoporus volvatus Extract Inhibits Influenza Virus Replication In Vitro and In Vivo

Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.     

The laboratory epidemiological study of the joint circulation of the influenza virus A subtypes A/H1N1/ and A/H3N2/ in Bulgaria]

The National Influenza Center of Bulgaria made the epidemiological analysis of the spread of influenza virus, type A, for the period of 11 years on the basis of mass laboratory investigations. Subtype A (H1N1) was found to be the main factor of epidemics in 1978 and 1982, while the epidemics of 1980, 1983, 1985, 1986, 1987 and 1988 were mainly caused by subtype A (H3N2). The data of laboratory and epidemiological studies indicated that after 20-year absence influenza virus A, subtype A (H1N1), was found again to circulate among the population of Bulgaria, and in 1978-1988 circulated simultaneously with the previous subtype A (H3N2). The simultaneous circulation of two subtypes of influenza virus was of great importance for the frequency, spread and duration of influenza epidemics.     

Broad-spectrum antiviral effect of Agrimonia pilosa extract on influenza viruses

Influenza virus continues to emerge and re-emerge, posing new threats for humans. Here we tested various Korean medicinal plant extracts for potential antiviral activity against influenza viruses. Among them, an extract of Agrimonia pilosa was shown to be highly effective against all three subtypes of human influenza viruses including H1N1 and H3N2 influenza A subtypes and influenza B virus. The EC₅₀ value against influenza A virus, as tested by the plaque reduction assay on MDCK cells, was 14–23 μg/ml. The extract also exhibited a virucidal effect at a concentration of 160–570 ng/ml against influenza A and B viruses when the viruses were treated with the extract prior to plaque assay. In addition, when tested in embryonated chicken eggs the extract exhibited a strong inhibitory effect in ovo on the H9N2 avian influenza virus at a concentration of 280 ng/ml. Quantitative RT-PCR analysis data showed that the extract, to some degree, suppressed viral RNA synthesis in MDCK cells. HI and inhibition of neuraminidase were observed only at high concentrations of the extract. And yet, the extract's antiviral activity required direct contact between it and the virus, suggesting that its antiviral action is mediated by the viral membrane, but does not involve the two major surface antigens, HA and NA, of the virus. The broad-spectrum antiviral activity of Agrimonia pilosa extract on various subtypes of influenza viruses merits further investigation as it may provide a means of managing avian influenza infections in poultry farms and potential avian-human transmission.     

Antiviral efficacy of Limonium densiflorum against HSV-1 and influenza viruses

Viral infections remain a major threat to humans and animals and there is a crucial need for new antiviral agents especially with the development of resistant viruses. Several Limonium genus members (Plumbaginacea) have been widely used in traditional medicine for the treatment of infections. In this study, we investigated the antiviral activities of different fractions after successive extraction (hexane, dichloromethane, ethanol and methanol) of the halophyte Limonium densiflorum against H1N1 influenza and HSV-1 herpes viruses. In addition, TLC phytochemicals of the shoot extracts were analyzed. All extracts were tested for their cytotoxicity using a fluorometric resazurin assay. The antiviral activity of extracts was tested using four modes of action: virucidal test, pretreatment of cells with samples before infection, attachment assay and plaque reduction test. A good antiviral activity was found with ethanol and methanol extracts. They were most potent in HSV-1 inhibition than H1N1 influenza virus. The most potent inhibition was observed with ethanol extract, and it exhibited high levels of virucidal activity against HSV-1 (IC₅₀ = 6 μg/mL). It inhibits the replication of the virus by 75% when added after penetration of the virus, and by 100% when added during the viral attachment. It protects MDCK cells against influenza virus by abolishing virus to entry into the host cell (IC₅₀ = 55 μg/mL). After attachment of influenza virus, the ethanol extract displayed an appreciable inhibition of virus replication (IC₅₀ = 193 μg/mL). Methanol extract showed a moderate antiviral capacity against both viruses. While dichloromethane has excellent antiherpes potential, results were inappropriate because it was toxic to Vero cells, hexane extract has no effect. TLC analysis of these extracts showed that flavonoids and saponins were the major classes of natural products found in the shoot extracts that may be responsible for these antiviral activities.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

甲3型流行性感冒病毒血凝素的pH特征

本文报告,pH9.6碳酸缓冲液对甲3型流感病毒的血凝滴度有明显降低作用,对甲1型和乙型仅有轻微影响,对甲2型的影响则介于两者之间。用不同pH的碳酸缓冲液、磷酸缓冲液及盐水等,测定甲3型流感病毒的血凝滴度,结果表明高pH对其有明显影响。分别具有甲3、甲2或甲1血凝素的重组株,在pH9.6碳酸缓冲液中,其血凝素的稳定性也和上述结果一样,即具有甲3血凝素的重组株,其血凝素对pH9.6碳酸缓冲液最敏感;甲1重组株的血凝素较稳定:而具有甲2血凝素的重组株则介于两者之间。利用此pH特征测定新分离的经血凝抑制试验鉴定为甲3型和乙型流感病毒,得到同样结果,因此有可能利用此pH特征对新分离的甲3型流感病毒进行初步鉴定。     

Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.     

Influenza H5N1 and H1N1 Virus Replication and Innate Immune Responses in Bronchial Epithelial Cells Are Influenced by the State of Differentiation

Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of α2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells'' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.     

A Cross-Sectional Study of Avian Influenza in One District of Guangzhou, 2013

Since Feb, 2013, more than 100 human beings had been infected with novel H7N9 avian influenza virus. As of May 2013, several H7N9 viruses had been found in retail live bird markets (LBMs) in Guangdong province of southern China where several human cases were confirmed later. However, the real avian influenza virus infection status especially H7N9 in Guangzhou remains unclear. Therefore, a cross-sectional study of avian influenza in commercial poultry farms, the wholesale LBM and retail LBMs in one district of Guangzhou was conducted from October to November, 2013. A total of 1505 cloacal and environmental samples from 52 commercial poultry farms, 1 wholesale LBM and 18 retail LBMs were collected and detected using real-time RT-PCR for type A, H7, H7N9 and H9 subtype avian influenza virus, respectively. Of all the flocks randomly sampled, 6 farms, 12 vendors of the wholesale LBM and 18 retail LBMs were type A avian influenza virus positive with 0, 3 and 11 positive for H9, respectively. The pooled prevalence and individual prevalence of type A avian influenza virus were 33.9% and 7.9% which for H9 subtype was 7.6% and 1.6%, respectively. None was H7 and H7N9 subtype virus positive. Different prevalence and prevalence ratio were found in different poultry species with partridges having the highest prevalence for both type A and H9 subtype avian influenza virus. Our results suggest that LBM may have a higher risk for sustaining and transmission of avian influenza virus than commercial poultry farms. The present study also indicates that different species may play different roles in the evolution and transmission of avian influenza virus. Therefore, risk-based surveillance and management measures should be conducted in future in this area.     

Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection

Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.     

Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata

The emergence of highly pathogenic influenza A virus strains, such as the new H1N1 swine influenza (novel influenza), represents a serious threat to global human health. During our course of an anti-influenza screening program on natural products, one new licochalcone G (1) and seven known (2-8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, the efficacy of oseltamivir with the presence of compound 3 (5 μM) was increased against H274Y neuraminidase. This evidence of synergistic effect makes this inhibitor to have a potential possibility for control of pandemic infection by oseltamivir-resistant influenza virus.     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Influenza activity in Czechoslovakia and the USSR, 1980-1985

Between 1980 and 1985, Czechoslovakia had experienced 4 and the USSR 3 major influenza outbreaks. Of the 3 epidemic outbreaks in the USSR, 2 were associated with influenza B virus (in the 1980/81 and 1983/84 seasons) and 1 with influenza A virus of the H3N2 subtype. In the USSR, influenza A (H1N1) virus never predominated as a cause of epidemic during the 5 years period. In Czechoslovakia, 2 epidemics (in the 1980/81 and 1983/84 seasons) were due to influenza A (H1N1) virus. The epidemic in the 1981/82 season had two waves of unequal heights and a mixed type B and subtype A (H3N2) etiology; a two-wave epidemic associated with isolates of influenza A (H1N1) and influenza B viruses was also recorded in the 1983/84 season. The influenza A (H3N2) epidemic in 1983 was of explosive character. All influenza viruses circulating in the two countries between 1980 and 1985 were of the same antigenic profile, but were isolated from the epidemics that occurred in different influenza seasons. The virological surveillance revealed strains of virus closely related to drift variants detected from outbreaks in 1977-1979 and the new variants A/Chile 1/83, A/Philippines 2/82, A/Caen 1/84 and B/USSR 100/83.