Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection

ABSTRACT: BACKGROUND: Although gene exchange is not likely to occur freely, reassortment between the H5N1 highlypathogenic avian influenza virus (HPAIV) and currently circulating human viruses is aserious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported toactivate the influenza replicon activity. METHODS: The replicon activities of PR8 and WSN strains (H1N1) of influenza containing PA fromHPAIV A/Cambodia/P0322095/2005 (H5N1) and the activity of the chimeric RNApolymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (CPA)was then reconstituted and its growth in cells and pathogenicity in mice examined. Theinterferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells werecompared with those of WSN-infected cells. RESULTS: The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and CPAreplicated better than WSN in cells. However, the multi-step growth of C-PA and itspathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, andcaspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cellsbut not in WSN-infected cells. CONCLUSIONS: Apoptosis and interferon were strongly induced early in C-PA infection, which protected theuninfected cells from expansion of viral infection. In this case, these classical host-virusinteractions contributed to the attenuation of this strongly replicating virus.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Conformational changes and fusion activity of influenza virus hemagglutinin of the H2 and H3 subtypes: effects of acid pretreatment.

Marked differences were observed between the H2 and H3 strains of influenza virus in their sensitivity to pretreatment at low pH. Whereas viral fusion and hemolysis mediated by influenza virus X:31 (H3 subtype) were inactivated by pretreatment of the virus at low pH, influenza virus A/Japan/305/57 (H2 subtype) retained those activities even after a 15-min incubation at pH 5.0 and 37 degrees C. Fusion with erythrocytes was measured by using the octadecylrhodamine-dequenching assay with both intact virions and CV-1 monkey kidney cells expressing hemagglutinin (HA) on the plasma membrane. To study the nature of the differences between the two strains, we examined the effects of low-pH treatment on the conformational change of HA by its susceptibility to protease digestion, exposure of the fusion peptide, and electron microscopy of unstained, frozen, hydrated virus. We found that the respective HA molecules from the two strains assumed different conformational states after exposure to low pH. The relationship between the conformation of HA and its fusogenic activity is discussed in the context of these experiments.     

Collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice.

Collagenous lectins (collectins) present in mammalian serum and pulmonary fluids bind to influenza virus and display antiviral activity in vitro, but their role in vivo has yet to be determined. We have used early and late isolates of H3N2 subtype influenza viruses that differ in their degree of glycosylation to examine the relationship between sensitivity to murine serum and pulmonary lectins in vitro and the ability of a virus to replicate in the respiratory tract of mice. A marked inverse correlation was found between these two parameters. Early H3 isolates (1968 to 1972) bear 7 potential glycosylation sites on hemagglutinin (HA), whereas later strains carry 9 or 10. Late isolates were shown to be much more sensitive than early strains to neutralization by the mouse serum mannose-binding lectin (MBL) and rat lung surfactant protein D (SP-D) and bound greater levels of these lectins in enzyme-linked immunosorbent assays and Western blot analyses. They also replicated very poorly in mouse lungs compared to the earlier strains. Growth in the lungs was greatly enhanced, however, if saccharide inhibitors of the collectins were included in the virus inoculum. The level of SP-D in bronchoalveolar lavage fluids increased on influenza virus infection. MBL was absent from lavage fluids of normal mice but could be detected in fluids from mice 3 days after infection with the virulent strain A/PR/8/34 (H1N1). The results implicate SP-D and possibly MBL as important components of the innate defense of the respiratory tract against influenza virus and indicate that the degree or pattern of glycosylation of a virus can be an important factor in its virulence.     

新甲型H1N1(2009)病毒的早期分子特征

摘要：【目的】本世纪首次流感大流行的病原属于甲型H1N1流感病毒,在遗传特性和抗原等方面都有别于人群中流行多年的季节性H1N1流感病毒。为了深入了解病毒的遗传特性,跟踪病毒的演化趋势,及时发现具有流行病学意义的变异株,本研究对早期分离的甲型H1N1(2009)病毒的分子特性进行了详细分析。【方法】通过GenBank的流感资源中心下载相关毒株的基因组信息, 序列分析采用DNAStar软件包的EditSeq和MegAlign比较与病毒致病性和宿主特异性相关的氨基酸变化情况。以A/California/07/2009（H1N1）作为新甲型H1N1(2009)的代表株进行详细的分子特征分析。【结果】A/California/07/2009不具备高致病性流感病毒的分子特征;病毒编码的11个蛋白大部分保留有猪流感病毒的分子特征,同时也具有一些禽和人流感病毒的特征;PB1-F2在11aa,57aa和87aa后发生断裂,具有古典猪H1N1和人H1N1双重特点,这是甲型H1N1（2009）病毒一个特有的分子特征。【结论】首次详细分析了新甲型H1N1（2009）病毒的分子特征。随着病毒在人群中的进一步适应和持续存在,这些分子特征将发生变化,应该特别关注这些变化对病毒的传播力和致病性的影响。     

Genomic peculiarity of coding sequences and metabolic potential of probiotic Escherichia coli strain Nissle 1917 inferred from raw genome data

Probiotic Escherichia coli strain Nissle 1917 (O6:K5:H1) is a commensal E. coli isolate that has a long tradition in medicine for the treatment of various intestinal disorders in humans. To elucidate the molecular basis of its probiotic nature, we started sequencing the genome of this organism with a whole-genome shotgun approach. A 7.8-fold coverage of the genomic sequence has been generated and is now in the finishing stage. To exploit the genome data as early as possible and to generate hypotheses for functional studies, the unfinished sequencing data were analyzed in this work using a new method [Sun, J., Zeng, A.P., 2004. IdentiCS--identification of coding sequence and in silico reconstruction of the metabolic network directly from unannotated low-coverage bacterial genome sequence. BMC Bioinformatics 5, 112] which is particularly suitable for the prediction of coding sequences (CDSs) from unannotated genome sequence. The CDSs predicted for E. coli Nissle 1917 were compared with those of all five other sequenced E. coli strains (E. coli K-12 MG1655, E. coli K-12 W3110, E. coli CFT073, EHEC O157:H7 EDL933 and EHEC O157:H7 Sakai) published to date. Five thousand one hundred and ninety-two CDSs were predicted for E. coli Nissle 1917, of which 1065 were assigned with enzyme EC numbers. The comparison of all predicted CDSs of E. coli Nissle 1917 to the other E. coli strains revealed 108 CDSs specific for this isolate. They are organized as four big genome islands and many other smaller gene clusters. Based on CDSs with EC numbers for enzymes, the potential metabolic network of Nissle 1917 was reconstructed and compared to those of the other five E. coli strains. Overall, the comparative genomic analysis sheds light on the genomic peculiarity of the probiotic E. coli strain Nissle 1917 and is helpful for designing further functional studies long before the sequencing project is completely finished.     

Primary structure of the full-length DNA copy of the influenza virus A/Kiev/59/79 (H1N1) PB2 gene

The complete nucleotide sequence of a cloned full-length DNA copy of the A/Kiev/59/79 (H1N1) influenza virus PB2 gene has been determined. This strain is shown to be the natural reassortant which inherited its NP and PB2 genes from the contemporary H3N2 influenza strains.     

应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。     

四株大肠埃希氏菌国际参考菌株动力和鞭毛抗原检定的研究

Authors identified four strains of Escherichia coli from Collaborative Centre for reference and research on Escherichia coli-Klebsiella (WHO) in Denmark. Our results were different from original record and reference. It was shown that strain H311b and H5 were motile strains, and its H antigen respectively is H11 and H27. The strain W27 is nonmotile. H antigen of the strain H511 is H40, not H8. Antigen formula of four strains respectively is 26:60:11 (strain H311b), 81:97:27 (strain H5), 115:-:- (strain W27), 102:-:40 (strain H511).     

甲型H1N1流感病毒感染不同免疫缺陷小鼠的比较

目的宿主免疫系统的功能状态在病毒的感染中起着至关重要的作用,本实验观察了不同免疫缺陷小鼠感染甲型H1N1流感病毒的差异。方法使用六个品系的近交系小鼠,经乙醚麻醉后进行滴鼻攻毒,分析其在病毒感染后存活率、体重变化和肺组织病理改变的异同。结果感染H1N1病毒的6种小鼠在观察的14d内,野生型的C57BL／6小鼠感染开始体重缓慢下降,感染后期有所回升,有半数存活;BALB／c小鼠和四种免疫缺陷品系小鼠感染病毒后体重随病情发展快速下降,死亡率均为100％。野生型C57BL／6小鼠感染初期为较弥漫的间质性肺炎,后期病变逐渐局限;BALB／c小鼠和四种免疫缺陷品系小鼠感染病毒后出现弥漫的中重度间质性肺炎,细支气管上皮有变性坏死,但炎症细胞明显少于C57BL／6小鼠。结论在甲型H1N1流感病毒的初次感染中固有免疫和特异性免疫分别在感染的初期和后期起主要作用,宿主免疫系统的功能状态影响着甲型H1N1病毒感染和预后。     

Pandemic Influenza H1N1 2009, Innate Immunity,and the Impact of Immunosenescence on Influenza Vaccine

Seasonal and pandemic strains of influenza have widespread implications for the global economy and global health. This has been highlighted recently as the epidemiologic characteristics for hospitalization and mortality for pandemic influenza H1N1 2009 are now emerging. While treatment with neuraminidase inhibitors are effective for seasonal and pandemic influenza, prevention of morbidity and mortality through effective vaccines requires a rigorous process of research and development. Vulnerable populations such as older adults (i.e., > age 65 years) suffer the greatest impact from seasonal influenza yet do not have a consistent seroprotective response to seasonal influenza vaccines due to a combination of factors. This short narrative review will highlight the emerging epidemiologic characteristics of pandemic H1N1 2009 and focus on immunosenescence, innate immune system responses to influenza virus infection and vaccination, and influenza vaccine responsiveness as it relates to seasonal and H1N1 pandemic influenza vaccines.     

Intracellular proteolytic cleavage of the influenza virus protein NP as a sign of the epidemicity of virus strains?

The main nucleocapsid protein (NP) of human epidemic viruses was found to be cleaved via NP56----HP53 mol. wt. reduction in infected cells, while the NP of animal influenza viruses was refractory to analogous intracellular modification. Like animal influenza viruses, the strain A/Baku/799/82(H1N3) isolated from a sick child has been observed to exhibit the intracellular resistance of NP to intracellular proteolysis. The similar NP resistance has been revealed for A/New Jersey/8/76(H1N1) and A/seal/Massachusetts/81 (H7N7) viruses, which are able to induce only a sporadic human influenza viral infection. Thus, the results reveal a correlation between the viral strains epidemicity and intracellular cleavability of their NPs. The influenza viral strains epidemic for humans are characterized by cleavable NP, whereas the strains, which are known to induce the sporadic influenza human infection are found to exhibit the resistance of NP to intracellular proteolysis. It is reasonable to consider the phenomenon of NP56----NP53 proteolytic modification as a sign of viral strain epidemicity for humans.     

Emergence of avian H1N1 influenza viruses in pigs in China.

Avian influenza A viruses from Asia are recognized as the source of genes that reassorted with human viral genes to generate the Asian/57 (H2N2) and Hong Kong/68 (H3N2) pandemic strains earlier in this century. Here we report the genetic analysis of avian influenza A H1N1 viruses recently isolated from pigs in southern China, a host suspected to generate new pandemic strains through gene reassortment events. Each of the eight gene segments was of avian origin. Phylogenetic analysis indicates that these genes form an Asian sublineage of the Eurasian avian lineage, suggesting that these viruses are an independent introduction into pigs in Asia. The presence of avian influenza viruses in pigs in China places them in an optimal position for transmission to humans and may serve as an early warning of the emergence of the next human influenza virus pandemic.     

The changing nature of avian influenza A virus (H5N1)

Highly pathogenic avian influenza A virus subtype H5N1 has been endemic in some bird species since its emergence in 1996 and its ecology, genetics and antigenic properties have continued to evolve. This has allowed diverse virus strains to emerge in endemic areas with altered receptor specificity, including a new H5 sublineage with enhanced binding affinity to the human-type receptor. The pandemic potential of H5N1 viruses is alarming and may be increasing. We review here the complex dynamics and changing nature of the H5N1 virus that may contribute to the emergence of pandemic strains.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.     

Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata

The regulation of the antioxidant defence system by ultraviolet-B (UV-B) was determined in a marine macroalga Ulva fasciata Delile exposed to low (0.5, 1 W m(-2)), medium (2.5, 5 W m(-2)), and high (10, 20 W m(-2)) UV-B irradiance. UV-B > or =2.5 W m(-2) increased H2O2 contents that are positively correlated with lipid peroxidation and total peroxide contents. Inhibition of the UV-B-induced H2O2 increase by a specific O2.- scavenger, 1,2-dihydroxy-benzene-3,5-disulphonic acid, shows that O2.- is the primary source of H2O2. Superoxide dismutase activity was increased by UV-B with a peak at 2.5 W m(-2), which did not match the H2O2 pattern. Alleviation of UV-B-induced oxidative damage by a H2O2 scavenger, dimethylthiourea, and a free radical scavenger, sodium benzoate, which inhibited UV-B-induced H2O2 accumulation, suggests that oxidative damage caused by UV-B > or = 2.5 W m(-2) is ascribed to accumulated H2O2. However, a decrease in growth rate and TTC reduction ability only at high UV-B doses indicates that the defence and repairing systems operate at low and medium UV-B doses. H2O2 not only can be excreted but can also be detoxified via the ascorbate-glutathione cycle. Increases in catalase, peroxidase, ascorbate peroxidase, and glutathione reductase activities and ascorbate (AsA) and glutathione pools, as well as AsA regeneration ability, function to keep the balance of cellular H2O2 under low UV-B doses. Dehydroascorbate reductase and monodehydroascorbate reductase are responsible for AsA regeneration under low and medium UV-B radiation, respectively. The appearance of oxidative damage in medium and high UV-B flux is attributable to a lower induction of the ascorbate-glutathione cycle as an antioxidant defence system. Overall, the availability of antioxidants and the induction of antioxidant enzyme activities for detoxifying reactive oxygen species (ROS) are regulated in U. fasciata against UV-B-induced oxidative stress, and experiments using ROS scavengers demonstrate that the antioxidant defence system is modulated by O2.- or H2O2.