Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses.Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses  and non-enveloped viruses . We found that extracts from persimmon ( Animal viruses, which are causative agents of human and animal diseases, can be divided into two types based on their physical properties: enveloped virus, which has a viral envelope composed of a lipid bilayer on its surface, and non-enveloped virus, which lacks an envelope with its protein shell usually being exposed to the environment Influenza virus, an enveloped virus, is a pathogen that causes respiratory infection. Outbreaks of influenza virus infection occur every year and sometimes cause fatal diseases including pneumonia, secondary bacterial pneumonia and encephalopathy Diospyros kaki), have been widely utilized for anti-septics, folk medicine and a clarifier for brewing Japanese sake. Green tea tannin, which is a hydrolyzable tannin, has been found to restrict the growth of viruses such as influenza virus and herpes simplex virus type 1 Tannins are basically derived from plants and defined as “a material which produces leather from hide” In the present study, we investigated the effects of seven extracts and chemical compounds containing tannins against twelve enveloped and non-enveloped viruses, and we found that persimmon extracts alone inactivated all of the viruses. We further tried to determine the mechanism by which persimmon extracts inactivate viruses.Persimmon extracts (PE) were prepared by serial procedures of squeezing immature green persimmon fruit, clarification with filtration, and freeze-drying. A batch of PE was shown to contain 21.8% of persimmon tannins . Pentagalloyl glucose (PGG) was prepared by crushing gallnuts followed by extraction with water, fractionation using an Octa Docyl Silyl column and freeze-drying. Wattle extracts (WE) from an acacia species, coffee extracts , propyl galate , and green tea extracts  were provided by Fuji Chemical Industry Co., Ltd. . Pyrogallol  was purchased from Kishida Chemical Co., Ltd. . These reagents were individually dissolved to make a 1% (w/w) solution in 10% (w/w) ethanol and kept at 4°C in a dark room as stock solutions. Tannin samples, their abbreviations, and the origins and types of tannin are summarized in 2 cells , Vero cells , MA104 cells  was added and the cells were further incubated for 4 h. The cells were solubilized and optical absorbance at 570 nm was measured by using a microtiter plate reader . Cell viability was estimated by comparing values of tannin samples with that of DMEM without tannins.Seven tannin samples were subjected to cytotoxicity assays by using a Cell Proliferation Kit I  according to the manufacturer's instructions. Briefly, confluent cells in a 96-well plate were exposed to 50 µl/well of DMEM containing 0.05%, 0.025% or 0.005% of tannins for 24 h in a CO50 method. Briefly, equal volumes of 0.5% tannin solution and virus solution were mixed to make a 0.25% tannin-containing virus solution. After 3-min incubation at room temperature, the solution was 10-fold serially diluted with DMEM. Susceptible cells for a virus in a 96-well plate were inoculated with 50 μl of a diluted virus solution in quadruplicate or octuplicate. After 1-h adsorption, the inoculum was removed and cells were incubated in 100 µl/well of DMEM supplemented with penicillin G and streptomycin. Twenty µg/ml of trypsin was included for the assay of influenza viruses, H3N2 and H5N3, and 5% FCS-containing MEM was used instead of DMEM for the assay of MNV. Water, Dulbecco's phosphate-buffered saline (PBS) and 10% ethanol were used instead of a tannin solution, and the average infectivity of the three was used as virus infectivity of mock-treated control. When the cytopathic effect had fully developed after several days, cells were fixed with ethanol and acetate (5∶1) and further stained with 0.5% amido black 10B in 45% ethanol and 10% acetate. The 50% endpoint of virus infection was determined by the Behrens-Kaeber method, and 50% tissue culture infectious dose (TCID50) was calculated. Anti-virus effects were estimated by comparing tannin-treated infectivity with mock-treated infectivity.Anti-virus assays for H3N2, H5N3, HSV, VSV, NDV, PoV, CoV, FCV and MNV were performed by a standard TCIDAnti-virus assays for SeV, AdV and RoV were performed by an immunofluorescent infectious focus assay. Briefly, tannin-treated and diluted virus solutions were inoculated to susceptible cells on a glass coverslip. After 24 h, cells were fixed and stained using a specific antibody as described previously 10 reduction or more (99.99% reduction or more) compared with mock-treated infectivity.Anti-virus effects were recognized when virus infectivity was decreased after tannin treatment by 4 Log50 assay. (1) MDCK cells in a 96-well plate were treated with 100 μl/well of DMEM containing 0.025% PE for 1 h before virus inoculation (pre-infection). (2) Virus solution was mixed with an equal volume of 0.5% PE and incubated for 3 min. After 10-fold serial dilution, 50 μl/well of the diluted virus inoculum was applied to cells in a well and kept at 37°C for 1 h as described above (adsorption). (3) After virus adsorption, the inoculum was replaced with 100 μl/well of DMEM containing 0.025% PE and 20 µg/ml of trypsin (post-infection). After virus infection in the three different conditions, virus infectivity was assessed as described above.PE was added in different steps of influenza virus infection in a standard TCID2 cells on a coverslip in a 35-mm dish were infected with SeV-EGFP at an input multiplicity of infection of 5 under PE treatment in three different conditions, (1) pre-infection, (2) adsorption and (3) post-infection, as described above. After 24 h, EGFP fluorescence was observed under a fluorescent microscope.Alternatively, monolayers of LLC-MKHI test was performed according to a standard procedure. Briefly, 25 μl of 0.1% PE or 0.1% bovine serum albumin (BSA) was 2-fold serially diluted in a round-bottom 96-well plate. Influenza virus H3N2 (16 HA in 25 μl) was added to each well and incubated at room temperature for 3 min. Then 0.5% (v/v) of chicken red blood cells in PBS (50 μl) was added to each well and hemagglutination was measured after 1 h.Influenza virus H3N2 particles were concentrated from infected allantoic fluid of chicken eggs by ultracentrifugation, followed by purification in a continuous sucrose gradient in ultracentrifugation and by suspension in PBS as described previously Accelerated aging was performed according to the guidelines. One percent (w/w) PE in 10% ethanol was kept for 0, 1, 4, or 6 months at 40°C±2°C and at relative humidity of 75%±5% and was subjected to anti-virus test against influenza virus.2, Vero, MA104, CRFK and RAW264.7 cells , FL, LLC-MK.7 cells , were in.7 cells , for 24 In the anti-viral test, 0.5% tannin sample was mixed with an equal volume of virus solution and then 10-fold serially diluted. Therefore, the highest concentration of tannin to which cells were exposed was 0.025%. We thus employed 0.025% tannin samples together with 0.05% and 0.005% samples.570) in the presence of 0.025% tannin samples compared with 0% tannin-, mock-treated individual cells  had lost most of its anti-virus activity . Next, w50 assay  with PE treatment in the three different conditions described above. After 24 h, EGFP expression was observed under a fluorescent microscope. A similar experiment was performed in SeV infection. LLC-MKTo further support the notion that PE inhibits virus attachment to cells, HI by PE was investigated. High concentrations (0.05% and 0.025%) of PE inhibited hemagglutination by influenza virus, while the same concentrations of BSA did not . These rWe next investigated interaction of PE with virions (virus particles). Purified virions of influenza virus H3N2 were incubated with PE at room temperature for 10 min, and viral proteins were analyzed by SDS-PAGE . Viral pTo confirm this notion, competition of BSA with PE in anti-virus effects was investigated. PE and different concentrations of BSA were incubated, and then the PE solution was subjected to an anti-virus test for influenza virus H3N2 . IncreasPolyphenols are strong anti-oxidants, and oxidation changes at least their color with time It is important for prevention of virus infection to inhibit transmission of pathogens. One of the means is the use of an anti-microbial reagent. However, some viruses, especially non-enveloped viruses such as human norovirus, have high levels of resistance to anti-microbial reagents such as detergents and ethanol. In the present study, we searched for an anti-viral reagent to efficiently inactivate both enveloped and non-enveloped viruses. We investigated plant-derived tannin samples, since catechins, tannins from green tea or black tea, were reported to inhibit pathogenic viruses such as influenza virus, herpes simplex virus type 1 and human immunodeficiency virus When PE was applied to cells before or after infection, it did not suppress virus infection. However, PE abrogated virus infection when it was applied in the period of virus adsorption. PE appeared to react with virus particles and inactivate virus attachment to cells. Inhibition of adsorption was reported in inactivation of herpes simplex virus by tannic acid by using radiolabeled virus particles SDS-PAGE analysis showed that PE induced aggregation of purified virions or BSA probably through association with proteins. Epigallocatechin gallate was previously shown to inactivate herpes simplex virus, and SDS-PAGE analysis demonstrated a complex formation of purified viral glycoproteins by the catechin Green tea extracts (GTE) contain hydrolyzable tannins and the main ingredients are (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechin-3-gallate and (−)-epigallocatechin-3-gallate In summary, the present study revealed that PE has inhibitory effects on the broadest range of viruses among the seven representative plant-derived tannin preparations. Investigation of the inhibitory mechanism of influenza virus and SeV suggested that persimmon tannins interacted with virion proteins and restricted virus adsorption to the cells. Due to the rapidly elicited anti-viral effect of persimmon tannins and their stability and safety, persimmon tannins can be utilized as anti-virus reagents to prevent virus infection. Especially, PE may be useful for inactivation of norovirus, which is a major cause of foodborne gastroenteritis, on hard surface.Figure S150 method.Effects of PE on virus attachment and virus invasion were separately investigated. (2a) To investigate the effect of PE treatment on virus attachment, influenza virus H3N2 was mixed with an equal volume of 0.5% PE and incubated for 3 min. After 10-fold serial dilution, 50 µl/well of the diluted virus inoculum was applied to pre-chilled cells and kept at 4°C for 1 h. Cells were then washed with 50 µl/well of pre-chilled PBS three times, incubated in 50 µl of pre-warmed DMEM and kept for 1 h at 37°C. (2b) To investigate the effect of PE treatment on virus invasion, influenza virus H3N2 was applied to pre-chilled cells MDCK cells and kept at 4°C for 1 h. Then the cells were then washed with 50 µl/well of pre-chilled PBS three times and supplemented with pre-chilled DMEM containing PE. The temperature was then increased to 37°C, which causes endocytosis and membrane fusion (virus invasion), and the temperature was maintained at 37°C for 1 h. In both cases, cells were further washed with PBS three times and incubated in 100 μl/well of DMEM containing 20 µg/ml trypsin, followed by infectivity measurement by the TCID(TIF)Click here for additional data file.Figure S2An equal volume of purified H3N2 virions (10 μg/μl) was mixed with a reagent as indicated in the figure and kept for 3 min. The samples were further mixed with an equal volume of 2× SDS sample buffer  and kept at 50°C for 20 min. Proteins were analyzed by 12% SDS-PAGE and Coomassie brilliant blue staining. Circle indicates proteins probably stacked at the bottom of a loading well of the PE sample.(TIF)Click here for additional data file.


Late diagnosis not only adversely affects individual health and survival but may also be associated with higher need for care, thus resulting in higher expenses for the healthcare system.In Austria up to 25% of newly diagnosed HIV infected patients present with less than 200 CD4 cell/mmTo assess the marginal costs of late presentation of HIV-infection in Austria.3 and never AIDS at presentation) presenting at the HIV-unit of the Medical University of Vienna between July 2006 and November 2008. Calculation of costs was based on official standard reimbursement systems for in-patient care, out-patient consultations, diagnostic procedures, and drug prices paid by the social insurance company or the hospital.Direct costs incurred during follow-up, demographic and clinical data were retrospectively collected for all late presenters (=cases) and an age- and risk-group matched cohort of controls . Median overall costs for late presenters incurred during the observation period were nearly 5 times higher for cases than for controls . This difference was driven by higher costs for out-patient consultations , in-patient care , diagnostic procedures  and ART . Costs for non-antiretroviral drugs did not differ significantly between the groups.Late presentation of HIV-infection is associated with a significant economic burden for the health system during the initial period of care. Higher costs for ART due to the immediate need for treatment account only for part of these costs. Interventions promoting earlier diagnosis of HIV in Austria may therefore prove to be cost-effective.


The production and consumption of energy and its derived services have raised the living standards of billions. Energy is traded in markets, and its price in most cases does not reflect the full cost of its use to society. Absent regulation, only the private cost of producing, for example, a kilowatt hour (kWh) of electricity is paid for by consumers through their electricity bills. These private costs, however, fail to account for a variety of external costs from this production activity in the form of, for example, present-day health damages from increased air pollution or potentially lower grain yields due to global warming halfway around the world and almost a century into the future. Economists argue that regulators should make producers “internalize” the external costs, preferably through flexible market mechanisms such as pollution taxes or cap-and-trade systems. Although qualitatively this can be easily explained to an undergraduate student in a 45-minute lecture, the exact magnitude of the full external costs is impossible to determine. The difficulty in determining the damages is caused by the significant degree of uncertainty in our understanding of the source-to-dose relationship combined with an even more limited understanding of the dose–response relationships for the vast variety of pollutants and potentially affected populations and systems. Monetizing the damages  is further complicated by the fact that many affected “receptors” of the pollution are not traded in markets and therefore do not carry a price.Hidden Costs of Energy, which provides quantitative, qualitative, and in some cases monetized estimates of these external damages from the use of energy for electricity, transportation, heat, and infrastructure and security.The National Academy of Sciences was charged by Congress to “define and evaluate the health, environmental, security, and infrastructural external costs and benefits that are not or may not be fully incorporated into the market price of energy [or] into the federal tax or fee.” The National Research Council committee, convened for this purpose and chaired by Jared Cohon and vice-chaired by Maureen Cropper, produced the report The central estimate of damages, which has been widely reported in the press, of US$120 billion for the last year of available data (2005) must be interpreted as a strict lower bound of the true external costs. Fifty-two percent of the estimated damages arises from health-related damages from the combustion of coal for electricity; 47% stems from the combustion of liquid fuels for transport. The remaining US$2 billion in damages are attributable to health effects from natural gas for electricity production and heating. To put this in perspective, the average price per kilowatt hour of electricity in the United States is approximately 10 cents—slightly higher for residential customers and slightly lower for industrial customers. The health damages from coal alone are 3.2 cents/kWh, with some plants having external costs at 12 cents/kWh.The book does not include the damages from climate change in its total costs, yet provides an intriguing calculation. If one assumed marginal damages to be $30/ton of carbon dioxide, the external costs from climate change per kilowatt hour are also in the neighborhood of 3 cents—doubling the estimated external costs.Two other factors that will drive up the true external costs of energy are the impacts on ecosystems and the impacts on national security—either of which are extremely difficult to value. For health damages, the report uses the value of a statistical (human) life of US$6 million, which is based on a large universe of existing willingness-to-pay studies. With the dearth of such studies in the heterogeneous ecosystem context, one cannot credibly and comprehensively determine what the overall monetary damages on ecosystems are.Even though a number of important effects have not been monetized, this report is the essential work in this area and belongs in the library of any serious researcher, policy maker, or writer working in this area. It is the most serious yet accessible assessment and literature review available of the external life-cycle costs of the energy system for the U.S. economy.


Although ceramic hydroxyapatite (HAp) chromatography has been used as an alternative method ultracentrifugation for the production of vaccines, the mechanism of virus separation is still obscure. In order to begin to understand the mechanisms of virus separation, HAp surfaces were observed by scanning electron microscopy after chromatography with dengue viruses. When these processes were performed without elution and with a 10–207 mM sodium phosphate buffer gradient elution, dengue viruses that were adsorbed to HAp were disproportionately located in the columns. However, when eluted with a 10–600 mM sodium phosphate buffer gradient, few viruses were observed on the HAp surface. After incubating the dengue viruses that were adsorbed on HAp beads at 37°C and 2°C, the sphericity of the dengue viruses were reduced with an increase in incubation temperature. These results suggested that dengue virus was adsorbed to the HAp surface by electronic interactions and could be eluted by high-salt concentration buffers, which are commonly used in protein purification. Furthermore, virus fusion was thought to occur with increasing temperature, which implied that virus-HAp adhesion was similar to virus-cell adhesion. The methodology of density-gradient ultracentrifugation, which has traditionally been applied in the purification of viruses, is able to achieve satisfactory results in vaccine production. However, the ultracentrifugation process is cumbersome and time-consuming In this study, in order to visually reveal some of the mechanisms underlying virus purification by HAp chromatography, we attempted to observe the location of adsorbed viruses with a scanning electron microscope (SEM) and count the number of dengue viruses that were adsorbed onto the HAp surface. Although viruses in column effluent have been analyzed previously, this study offers a new perspective that would help to understand viral behaviors inside a chromatographic column.Furthermore, the dengue virus morphologies on the HAp surface after the incubation processes were observed by SEM in order to understand the virus-HAp interactions. The dengue virus is a member of the flavivirus family that carries a single-stranded RNA with an envelope and has a virus diameter of approximately 50–60 nm Aedes albopictus mosquito C6/36 cells Dengue virus strain type 2 ThNH7/93 and l-glutamine  that was supplemented with 10% fetal calf serum.The cell culture medium used was Eagle’s Minimum Essential Medium with l-glutamine  supplemented with 0.5% fetal calf serum.The virus production medium used was Autopow- Eagle’s Minimum Essential Medium without 2 culture flask that was precoated with 100 µg/mL of poly-l-lysine  at 28°C for 7 days in order to reach confluence. The dengue virus was then inoculated onto the cells in 75 mL of the virus production medium. After culturing for 3 days, the medium was changed with 75 mL of fresh medium and then cultured at 28°C for a further 4 days . We subsequently collected 75 mL of the 7-day culture fluid. The dengue virus culture fluid was filtered through a 0.22-µm pore-size membrane.C6/36 cells were grown in 45 mL of the cell culture medium in a 225-cmHAp suspension (Ca/P = 1.67) was prepared using calcium hydroxide and phosphoric acid. The suspension was dried to obtain spherical particles  by using a spray-dry technique, and then, the particles were sintered at 950°C. The particles were packed into an empty stainless-steel column . Three columns  were prepared for use with 3 different elution processes, as described in the Materials and methods, section 5. Another 3 columns  were prepared for incubation under 3 different conditions, as described in the Materials and methods, section 6.Dengue virus adsorption to Columns A–F was performed using the BioLogic chromatography system . Virus culture fluid (25 mL) was loaded onto each column for adsorption, and the columns were equilibrated with 45 column volumes (CV) of 10 mM sodium phosphate buffer (NaPB), pH 7.2. The flow rate was adjusted throughout at 1 mL/min. The effluents were monitored at 280 nm and 260 nm. The fractions were collected every minute.Dengue viruses were similarly adsorbed to Columns A–C, as described in the Materials and methods, section 4, as follows: Column A, no elution was performed; Column B was eluted with a 10–207 mM NaPB (pH 7.2) gradient (11 CV); and Column C was eluted with a 10–600 mM NaPB (pH 7.2) gradient (34 CV). The flow rate, monitoring of the effluents, and collection of the fractions were conducted under the same conditions as described in the Materials and methods, section 4. There were no time lags between the virus adsorption processes described in the Materials and methods, section 4, and the elution processes described in the Materials and methods, section 5.Dengue viruses were similarly adsorbed onto Columns D–F, as described in the Materials and methods, section 4. Column D was not incubated, Column E was incubated at 2°C for 7 days, and Column F was incubated at 37°C for 7 days.After each process, Columns A–F were unpacked by pushing the column out with a syringe while being careful not to mix the HAp particles of the different parts of the column. HAp particles that were unpacked from Columns A–C were collected from the 3 sections of each column: the column head, the mid-column, and the column end .However, HAp particles that were unpacked from Columns D–F were collected only from the column head. Each collected particle was fixed with 1% glutaraldehyde  in 100 mM NaPB (pH 7.4), for 30 min and washed 3 times in 100 mM NaPB (pH 7.4). The particles were further fixed with 1% osmium (VIII) oxide  in 100 mM NaPB (pH 7.4), for 30 min and washed 3 times in 10 mM NaPB (pH 7.4).Each HAp particle in 10 mM NaPB (pH 7.4), was mounted onto a metal plate with carbon tape and dried at room temperature. The particles were coated with platinum-palladium in an E-1030 Ion Sputter  and observed at 8.0 kV or 10 kV with an S-4200 or S-4300 scanning electron microscope (Hitachi High-Technologies Corporation). The process flow chart of the elutions and incubations, as described in the Materials and methods, sections 3–7, are shown in The viruses adsorbed on HAp were identified as dengue viruses if they were of a particle diameter within the range 40–60 nm and if their shape was spherical. The sphericity of the virus particle was determined by calculating the difference between 2 orthogonal diameter measurements; if this difference was less than 20%, the viruses were classified as spherical, and if the difference was more than 20%, they were classified as flat.Goose red blood cells  were washed with phosphate-buffered saline (PBS) and suspended in PBS at a final concentration of 8% as a stock solution. This stock solution was diluted to 1∶24 in order to prepare a 0.33% goose red blood cell working suspension in virus-adjusting diluent containing 150 mM sodium chloride and 200 mM NaPB with different pH values . The optimal pH was selected to show the highest titer among them. The virus sample (50 µL) was diluted 2-fold serially with 0.4% bovine serum albumin in BS9 . Fifty microliters of the 0.33% red blood cell suspension was then added to each well, the plate was mixed gently, and it was incubated at 37°C for 30 min. The HA titer that was the highest dilution of virus showed the agglutination pattern on the bottom of the well.Dengue viruses were adsorbed to HAp particles in Columns A–C, and a different elution process was performed in each column. As shown in t test).t test), indicating the occurrence of a virus drift.For the Column B condition (elution with a 10–207 mM NaPB gradient), the numbers of adsorbed dengue viruses on the HAp surface of the column head and mid-column sections were nearly equivalent. However, the number of dengue viruses that were adsorbed onto the HAp surface of the column end section was quite small. We assumed that, at the beginning of the NaPB gradient, some viruses were desorbed by the low NaPB, and the viruses then moved to the outlet side of the column. Eventually, the drifting viruses were again adsorbed to downstream HAp particles. Statistical analysis also showed a significant difference between the column heads of Column A and Column B , only 2 dengue viruses were observed on the HAp surface of the column head section, and no dengue viruses were observed on the HAp surface of the column end and mid-column sections. As shown in Accordingly, the following findings of virus behaviors are visualized in t test analysis also showed significant differences between Column E and Column F (P<.01), between Column E and Column D (P<.01), and between Column D and Column F (P<.1). The shapes of the dengue viruses that were adsorbed to the HAp surface were altered from spherical to flat shapes with increasing temperature.The HAp chromatograms showed that the dengue viruses were separable from admixture proteins with HAp chromatography as well as with commonly used protein purification methods. However, concerning the width of the peaks, the C-1 UV peak for eluting admixture proteins was narrower than the C-2 UV peak for eluting dengue viruses . The denThe SEM images showed the in-column circumstances of the viruses on the HAp surface of the NaPB gradient from the start to the end . The debWhen comparing the chromatogram and SEM images, when HA units were not detected in any of the fractions, viruses could be observed in the corresponding SEM images. The opposite is shown in Dengue virus infections have increased in recent decades. Thus, vaccine development has also been active


Pneumovirinae within the family Paramyxoviridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups based on genetic or antigenic differences or both. For HMPV, the existence of different genetic lineages has been described on the basis of variation in a limited set of available sequences. We address the antigenic relationship between genetic lineages in virus neutralization assays. In addition, we analyzed the genetic diversity of HMPV by phylogenetic analysis of sequences obtained for part of the fusion protein (n = 84) and the complete attachment protein open reading frames (n = 35). On the basis of sequence diversity between attachment protein genes and the differences in virus neutralization titers, two HMPV serotypes were defined. Each serotype could be divided into two genetic lineages, but these did not reflect major antigenic differences.Human metapneumovirus (HMPV) is a member of the subfamily  Pneumovirinae subfamily within the Paramyxoviridae family (4). The Pneumovirinae subfamily consists of two genera: the pneumoviruses and the metapneumoviruses. Human respiratory syncytial virus (HRSV), the major viral cause of severe respiratory tract illnesses in children, is the type species of the pneumoviruses (Metapneumovirus genus until the discovery of HMPV (Human metapneumovirus (HMPV) has recently been identified as a causative agent of respiratory tract illnesses in humans worldwide  and the complete G open reading frames (ORFs) (n = 35). In addition, we addressed the antigenic relationship between the different lineages with virus neutralization assays using lineage-specific antisera raised in ferrets. Virologic studies have used a definition of a homologous-to-heterologous virus neutralization titer ratio of >16 as a definition for serotypes .Viruses were isolated on tertiary monkey kidney (tMK) cells as previously described  or at days 0 and 21 (ferret 3 to 6). Infections were performed as follows: ferrets 1 and 3: HMPV NL/1/00, prototype virus for lineage A1. Ferrets 2 and 5: HMPV NL/1/99, prototype virus for lineage B1. Ferret 4: HMPV NL/17/00, prototype virus for lineage A2 and ferret 6: HMPV UK/5/01 a virus from lineage B2. Ferrets were housed in isolator cages to avoid cross-infections.Lineage-specific polyclonal HMPV antisera were raised by infecting ferrets with 1 mL of virus-infected tMK supernatants containing approximately 10HMPV-specific polyclonal antisera were raised in guinea pigs as previously described . Each experiment included virus titrations of the working solution of the virus, using twofold dilutions, and 10–100 TCID50 per well was considered acceptable.Virus neutralization assays of heat-inactivated (30 min 56°C) ferret serum samples were performed as previously described  were obtained from clinical samples collected from 84 HMPV-infected patients. Phylogenetic analysis of these sequences confirmed two main genetic lineages, A and B. Each of these lineages appeared to consist of two sublineages, which were tentatively named A1, A2, B1, and B2 .Comparison of the sequences showed high percentage identities between members of the same sublineage , members of the two different sublineages within each main lineage , and between members of the two different main lineages A and B . Whereas no specific amino acid residue substitutions could be found between sequences from subgroups A1 and A2, there were 5 specific aa substitutions between sequences from genotypes A and B, and one substitution between B1 and B2 . The lowNucleotide sequences of the region between the start codons of the G and the polymerase (L) reading frame were obtained for 35 samples. Phylogenetic analysis showed the same clustering of the sequences over the four sublineages as seen for the F protein gene . The G rN-linked glycosylation sites varied even within each sublineage, from two to six potential sites, with one located at the proposed cytoplasmic tail conserved among all lineages.The position of the hydrophobic domain, a high percentage of proline, serine, and threonine residues and a cysteine residue at position 27 are features shared by all HMPVs. Whereas the cytoplasmic tail was conserved among all members (58%–70% aa identity), the proposed ectodomains (start aa 51) were quite variable (18%–25% aa identity between lineage A and B). The number and position of potential sites for Analysis of HMPV sequences obtained from samples received from different countries indicated that sequences from Finland, the United Kingdom, and the limited sequences from Asia and South America, were found on branches between the Dutch sequences in the F tree, and not as a separate lineage. The variation between sequences obtained from samples from a single country was found in the same range as the variation found between samples obtained from different countries. In agreement with the genetic lineages of HMPV observed worldwide, which usually includes sequences similar to those of isolate NL/1/00 or NL/1/99, geographic clustering does not appear to apply to HMPV.The different HMPV samples were obtained during the last 20 years with most from 2000 to 2002 and 14 in the 1990s. As indicator for possible fixation of amino acid variation over time, we analyzed the G ORF amino acid sequence of members in sublineage A2 and B1 (containing samples from 1981 to 2002) in more detail. The amino acid sequence variation between the viruses from 1981 and 2001 was in the same range as the variation found between viruses from 2001, and alignments of the sequences did not indicate fixation of amino acid changes between 1981 and 2002. Thus, antigenic drift, as observed for influenza A and B viruses, does not appear to be an important phenomenon for HMPV.50; this variation did not affect the measured virus neutralization titers , complete F (n = 4), and the complete G (n = 35) protein genes. Phylogenetic analysis of these sequences showed two main lineages (A and B) with each divided into two sublineages (1 and 2). As was described for HRSV and APV, the F protein was highly conserved, which is in agreement with F proteins of pneumoviruses having structural and functional constraints for amino acid mutations  were antigenically closely related. The difference in homologous and heterologous virus neutralization titers between members of the two different lineages A and B titers (12- to 128-fold higher homologous titer than heterologous titer) indicate a difference in antigenicity between lineage A and B. Classic virology studies have used a definition of a homologous-to-heterologous virus neutralization titer ratio of >16 for defining serotypes. This same definition notes that if neutralization shows a certain degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous titer ratio of 8 or 16), distinctiveness of serotype is assumed if substantial differences in sequences are observed . So far, one heterologous reinfection has been reported in humans (Our data support a technical description of two serotypes of HMPV in experimentally infected ferrets. The existence and relevance of these serotypes in other animal species, including humans, has yet to be determined.Our results in combination with data published by others (


Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways.von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y Under normal conditions, vWF does not interact with circulating platelets In vivo, von Willebrand Factor (vWF) interacts with platelet glycoprotein Ib complex (GPIb) under high shear and triggers platelet activation, which leads to ADP and TxAThe antibiotic ristocetin binds to the C-terminal part of the A1-domain of vWF as well as to GPIb, inducing the interaction between vWF and GPIb on platelets Echicetin, a snake venom C-type lectin, specifically binds to GPIb, but does not induce activation of washed platelets. Moreover, binding of echicetin to GPIb completely blocks vWF/R induced platelet agglutination Blood was obtained from healthy volunteers, who gave written informed consent according to our institutional guidelines and the Declaration of Helsinki. Our studies with human platelets and the consent procedure were approved and reconfirmed  by the local ethics committee of the University of Wuerzburg (Studies No. 67/92 and 114/04).Echis carinatus sochureki venom was from Latoxan . Ristocetin was from Biopool . vWF  was from CSL Behring . Polystyrene beads  and phospho–p38 (Thr180/Tyr182) MAPK antibody were from Sigma . R-phycoerythrin (RPE)-conjugated anti-CD62P antibody was from DAKO . Anti-phosphotyrosine antibody (4 G10) was from Millipore . Phospho-VASP Ser239, and phospho-ERK (Thr201/Tyr204) antibodies were from Nanotools . Antibodies against phospho-PKB (Thr308), VASP, and p38 were from Cell Signaling .Echicetin was purified as previously described 125 using Pierce Iodination Reagent (Iodogen) with a standard protocol . Unreacted I125 was removed from the labeled protein by gel-filtration on Sephadex G10. Labeled echicetin (10 μg) was mixed with 1 mg of unlabeled echicetin. This mixture was used as a stock to prepare 0.3 (EP0.3), 0.2 (EP0.2), 0.1 (EP0.1) and 0.05 mg/ml (EP0.05) echicetin solutions for adsorption on polystyrene beads using the same protocol as with unlabeled protein. The total radioactivity of the samples was measured before adsorption and the residual radioactivity on polystyrene beads was measured after adsorption using a gamma-counter. The amount of adsorbed echicetin was calculated from the ratio between the total sample radioactivity and the residual radioactivity on the polystyrene beads. We found that 28.2, 23.7, 13.2 and 7.5 μg of echicetin (30.6 kDa) bound to EP0.3, EP0.2, EP0.1 and EP0.05, respectively. Absorbed echicetin (A) corresponds to 5.7×1014, 4.8×1014, 2.7×1014, and 1.5×1014 molecules. To find the average distance between two neighboring echicetin molecules absorbed on polystyrene beads the total area of all beads in the suspension was calculated as follows.bead is the surface area of a single bead (0.6644 μm2 for a bead with d = 0.46 μm) and N is the total number of beads in the sample, calculated using an equation supplied by Sigma and determined to be 1.877×1010.Echicetin was labeled with Isingle is the area containing a single molecule, Stotal is the total area of polystyrene beads covered by echicetin and A is the number of adsorbed echicetin molecules. For EP0.3, EP0.2, EP0.1 and EP0.05, Ssingle was found to be 21.8, 25.7, 46.2 and 81.2 nm2 respectively. The distance between two neighboring molecules (D) was calculated as Assuming that echicetin is evenly distributed on the polystyrene surface, the mean area on the bead occupied by each echicetin molecule was calculated by the equation: 8/ml) were treated with echicetin beads (1% w/w suspension) or vWF/R under continuous stirring at 1000 rpm and 37°C.Human platelets were prepared as previously described Platelets activated with echicetin beads were taken directly from the aggregation cuvette and fixed with 1% formaldehyde for 10 min. After centrifugation, platelets were resuspended in PBS/0.5% BSA, and incubated with RPE-conjugated anti-CD62P antibody. FACS analysis was performed on a Becton Dickinson FACScalibur using CELLQuest software, version 3.1f .Cover slips were coated with various concentrations of echicetin. Platelet adhesion and spreading on echicetin coated surfaces were performed as described before For Western blot analysis, 2× Laemmli buffer was directly added to washed platelets stimulated with echicetin beads or vWF/R. Platelet proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and the membranes were incubated with appropriate primary antibodies. Goat anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase were used as secondary antibodies, followed by ECL detection.All experiments were performed at least three times and the data are expressed as mean ± SEM. Differences between groups were analyzed by ANOVA followed by Bonferroni’s test and Student t-test was used when appropriate. P<0.05 was considered statistically significant.Elucidation of intracellular signaling mechanisms induced by vWF/R in washed platelet suspension is limited by strong platelet agglutination. In PRP, addition of ristocetin induces two waves of platelet activation; first one is agglutination, and the second αIIbβ3-dependent aggregation . Therefo125 labeled echicetin on polystyrene beads (see methods). First, we established the range of echicetin concentrations (between 1 and 0.01 mg/ml) for coating the beads and found that maximum aggregation was induced by incubation with 0.3 mg/ml, whereas 0.05 mg/ml did not induce any significant platelet aggregation. According to our calculations (see methods) the number of echicetin molecules per bead should be 30500, 25800, 14300 and 8170 in EP0.3, EP0.2, EP0.1, and EP0.05, respectively and the average distance between the adsorbed echicetin molecules is 4.7, 5.1, 6.8 and 9.0 nm. Correspondingly, the total number of echicetin molecules added to platelet samples were 5.7×1014, 4.8×1014, 2.7×1014, and 1.5×1014 for EP0.3, EP0.2, EP0.1, and EP0.05, respectively. There are about 40000 molecules of GPIb per platelet 7 platelets used in aggregation experiments were 1.84×1012 approximately, which is comparable with the total number of echicetin molecules in PE0.05.Neither monomeric echicetin nor BSA coated beads (used as controls) induce platelet aggregation or agglutination . To eluc14 echicetin molecules per sample) and still found no significant changes in platelet aggregation  was observed with EP0.1 . Howeverregation . These dPlatelet activation induced by echicetin beads was determined by P-selectin expression on the activated platelets. Though thrombin and echicetin beads induce full platelet aggregation, P-selectin expression induced by echicetin beads was significantly less than in thrombin-stimulated platelets. There were no significant differences between P-selectin expression levels induced by EP0.3 and EP0.2. P-selectin expression was slightly lower with EP0.1 and strongly decreased in the case of EP0.05 . These dTo identify whether GPIb-mediated platelet activation induces platelet adhesion and spreading, washed human platelets were incubated on cover slips coated with increasing concentrations of echicetin. We found typical platelet adhesion and spreading on echicetin coated surfaces and the number of adherent platelets increased with the density of echicetin coated on the surface . The areGlobal protein tyrosine phosphorylation is a reliable marker of activation dependent intracellular signaling. Therefore, we examined changes in protein tyrosine phosphorylation in platelets activated by EP0.1. Strongly increased tyrosine phosphorylation of several proteins was observed in platelets stimulated by EP0.1, which was partially inhibited by aggrastat . This inPlatelets preincubated with Src-kinase inhibitor (PP2) did not form aggregates, when stimulated with EP. PP3, an inactive analog of PP2 did not affect EP-induced platelet aggregation . In cont2 play significant roles in shear-induced GPIb mediated platelet aggregation 2 for aggregation 2 signaling in EP-induced platelet aggregation, platelets were pre-incubated with antagonists of P2Y12 (AR-C69931), P2Y1 (MRS-2179) or TxA2 receptor (SQ-29548). AR-C69931 potently inhibits EP-induced platelet aggregation, whereas MRS-2179 had only a minimal effect. SQ-29548 strongly reduced EP-induced platelet aggregation  indicating that other intracellular mechanisms are involved in EP-induced platelet aggregation. Pre-treatment with AR-C69931 and SQ-29548 inhibits phosphorylation of PKB, ERK and p38 , and A1 domain which binds GPIb IIbβ3) induced by vWF/R treatment has been described in several papers 2 receptors , D’-D3 domain (possible binding site for P-selectin), A3 domain (the binding site for fibrillar collagen type I and III), C1 domain  that activates platelets via clustering of GPIb without directly involving other receptors. This should allow the dissection of the complex mechanisms involved in vWF-induced platelet activation. We show that the distance (less than 7 nm) between GPIb ligands rather than their concentration is critical for platelet activation/aggregation. We propose that echicetin-coated beads can be a powerful and reliable tool to study specific GPIb-dependent signaling pathways in platelets.


Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses – non-enveloped, icosahedral viruses remains unknown.We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation.We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future. Potential contamination of pharmaceuticals, biologicals, and uncooked foods with viruses is a critical problem. Conventional disinfection methods have potential serious side effects; for example, biochemical and pharmaceutical disinfection methods involve addition of potentially toxic or carcinogenic chemicals, such as detergents and photosensitizers which are impossible to remove completely after the treatments. In addition, the added chemicals may interact with the product itself, potentially altering its structure or function. Ionizing radiation such as ultraviolet and gamma rays can be used to sanitize foods and destroy viral pathogens in biologicals but amount of irradiation required for viruses is relatively high, potentially damaging biological products and cells and is less desirable for foods due to consumer concern. Microwave absorption method is not viable because water usually coexists with biological systems and water severely absorbs light in the microwave spectral range, leading to the heating effects.An ultrashort pulsed (USP) laser technology has recently been developed to circumvent these difficulties -10. The Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown -10 to efThere are a variety of possible inactivation mechanisms for the non-enveloped, icosahedral viruses. These possibilities include: (i) inactivation due to ultraviolet C (UVC) photons produced by the intense laser beam through non-linear optical process; (ii) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (iii) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (iv) inactivation via an ISRS process.We note that there is one more possibility of inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle , leadingIn this paper, we report examination of these possible inactivation mechanisms for a non-enveloped, icosahedral virus, namely murine norovirus-1 (MNV-1), and present evidence that supports ISRS as the most likely inactivation mechanism by a visible USP laser. MNV-1 was chosen in this study because norovirus, which is highly contagious, is one of the leading viruses for food poisoning around the globe. This efficient, non-invasive approach for the eradication of non-enveloped, icosahedral viruses, when employed in a continuous, syringe-pumped configuration, can be applied to the disinfection of pathogens in the pharmaceutical processes and disinfection of blood components for blood transfusion.g for 6 h at 4°C in a TH-660 rotor . The pellet was resuspended in PBS and further purified by centrifugation at 175,000 × g for 6 h at 4°C using a sucrose step gradient of 10, 20, 30, 40 and 45% sucrose. The pellet was resuspended in Earle’s balanced salt solution (Gibco-Invitrogen) and dialyzed against Tris-buffer saline .Working stocks of MNV-1 were prepared using confluent monolayers of mouse monocyte/macrophage RAW 264.7 cells  cultured in high glucose Dulbecco’s modified eagle media  without indicator supplemented with 25 mM HEPES buffer, 10% fetal bovine serum , 2 mM Gluta-MAX-1 (Gibco-Invitrogen), 100 U of penicillin, and 100 μg/ml of streptomycin sulfate (Gibco-Invitrogen), essentially as described by Wobus et al. . PartialMNV-1 samples were assayed using confluent 6-well dishes  inoculated with 0.5 ml, or ten-fold serial dilutions prepared in Earle’s balanced salt solution , for 2 h at 37°C followed by overlay with 2 ml of modified eagle media (Gibco-Invitrogen) containing 1.5% low melt agarose (Fisher Biotech) with 5% FBS, 2 mM Gluta-MAX-1, 100 U of penicillin and 100 μg/ml of streptomycin sulfate (Gibco-Invitrogen). After three days incubation, plaques were visualized by staining with 0.03% neutral red (Fisher Biotech) for 2 h at 37°C.3, 1.6 × 102, 1.6 × 101, 1.6, and 1.6 × 10-1.Because the 96-well tissue culture-treated flat bottom plates contain approximately 30,000 cells/well, depending upon the dilution of viral titer in the assaying process, the ratio of virus to cell can be 1.6 × 107 PFU/ml. The assays were performed on the laser-irradiated samples after proper dilution. The typical exposure time of the sample to laser irradiation was about 2 h. All the experimental results reported here were obtained at T = 25°C and with the single-laser-beam excitation. Temperature increase of sample solutions during USP laser treatments, as monitored by a thermocouple, did not exceed 2°C. The tightest focused spot of the laser beam was approximately 100 μm and the average laser exposure time of individual virion within the beam is estimated to be about 3.5 s.The excitation source employed in this work was a diode-pumped mode-locked Ti-sapphire laser. The laser produced a continuous train of 65 fs pulses at a repetition rate of 80 MHz. The output of the second harmonic generation (SHG) system of the Ti-sapphire laser was used to irradiate the sample. The excitation laser was chosen to operate at a wavelength of 425 nm and with an average power as specified. It had a pulse width of full-width at half maximum (FWHM) of about 100 fs. An achromatic lens of focus length 5 cm was used to focus the laser beam into the sample area. In order to facilitate the interaction of laser with MNV-1, the viral sample with a volume of 0.1 ml in buffer solution was placed inside a Pyrex cuvette with a micromagnet stirring bar and stationed above a magnetic stirrer so that virions would be forced to enter the laser-focused volume. The titer of MNV-1 samples was 5 × 10All the experiments were carried out in triplicate. The errors were expressed in standard deviations (SD). Temperature was measured during laser-irradiation experiments with a thermocouple.MNV-1 samples were visualized using 400 mesh, copper grids  coated with a 0.25% Formvar solution . Grids negatively stained with 1% phosphotungstic acid , pH 7.0 and examined under a Philips CM12 transmission electron microscope  at an accelerating voltage of 80 KV. Images were collected with a 4000 M-T1-GE-AMT detector  and processed with AMT V600 software .2, following the standard procedures as described by the manufacturer, by using Applied Biosystems model 7500 fast Real-Time PCR system from life Technologies. For MNV-1, sense primer 6622 s 5′-cgcctttaccaattggcc-3′ and antisense primer 6875 5′-tgaaagagttggtttggagc-3′ were used at an annealing temperature of 64°C to produce a 273-bp amplicon [Real-Time PCR measurements were performed on the genome of MNV-1 without the laser irradiation (control) and after the laser irradiation at an average laser power density of 100 MW/cmamplicon .Reverse transcription of all viral RNA was performed at 50°C for 30 min followed by a 15-min Taq activation step at 95°C. Forty cycle PCR reactions were performed using annealing times of 1 min, a 1-min extension step at 72°C and a 30-s denaturation step at 95°C. For the final cycle, the annealing time was extended to 2 min and the final extension was performed for 10 min. All primers were used at a final concentration of 0.1 μg/50 μl reaction mix or approximately 0.25 μM for each primer.10 scale for the laser-treated MNV-1 group. Here, load reduction is defined as the ratio of the number of plaque in the control to the number of plaque in the laser-irradiated sample. For reference, Figure 10 scale for the unpurified laser-treated MNV-1.Since virus stocks and samples are often propagated in complex tissue culture media that contains potentially chromogenic constituents such as neutral red indicator dye, amino acids, cellular proteins and nucleic acids, MNV-1 used in this work was propagated in indicator-free media and partially purified by sucrose gradient or Optiprep gradient ultracentrifugation, respectively. Figure We observed that the temperature of laser-treated samples did not increase by more than 2°C above room temperature as monitored by a thermocouple.2. As the laser power density increases beyond 80 MW/cm2, the fraction of survival drops precipitously.To gain better insight into the mechanism of inactivation for these non-enveloped, icosahedral viruses by USP laser irradiation, we measured the fraction of MNV-1 survival as a function of laser power density, which is shown in Figure MW/cm2, the capsid of the inactivated MNV-1 becomes cracked, presumably along the weak structural links, but remains intact, as evidenced by the appearance of smaller structures of about 10 nm in diameter on the capsid . This transmission electron microscope image clearly reveals the locations of weak structural links on the capsid of a non-enveloped, icosahedral virus – MNV-1. Figure MW/cm2, the capsid of the inactivated MNV-1 becomes disintegrated and separated into small pieces of diameter about 10 nm.Typical transmission electron micrographs of the control and laser-irradiated MNV-1 samples are shown in Figure -1, while substantial inactivation was observed for laser spectral width ≥ 2 cm-1.The theory of ISRS developed for the single-pulse excitation configuration makes predictions that the energy contained within the spectral width of a USP laser must be larger than the energy of a given molecular vibration to excite that specific vibrational motion in the molecule . In orde2. The similarity of real-time PCR counts between the control and laser-irradiated samples suggests that, within the amplicon size of 273 bp tested, there is very minimal genome degradation/damage for MNV-1 after the laser irradiation. This result is consistent with our previous reports by using gel electrophoresis that USP laser irradiation under similar experimental conditions does not damage the genome of either M13 bacteriophage [In order to test the effects of USP laser irradiation on the genome of MNV-1, Figure riophage ,8 or MCMriophage .As mentioned previously, potential mechanisms for the inactivation of non-enveloped, icosahedral viruses such as MNV-1 include: (i) production of UVC photons by non-linear optical process; (ii) thermal heating by the direct laser absorption; (iii) one-photon absorption process by chromophores, such as porphyrin molecules  or indicFirst, we have estimated the number of UVC photons which might be produced at laser power densities used in our experiments. The non-linear optical coefficient for murine norovirus is not available in the literature; however, if we assume that the viruses are giant molecules and that their non-linear coefficients are comparable to that of a typical molecule , then thSecondly, we have tried to detect the UVC photons which might be generated in our laser experiments  by using a UV spectrometer with a photon counting system. Within our experimental uncertainty of ±1photon per second we failed to detect any of the UVC photons. This finding is consistent with our estimation stated above. Therefore, the observed inactivation for MNV-1 cannot be due to UVC photons generated by non-linear optical process.In order for the USP laser to thermally damage the virus, there must be direct energy transfer between the laser and viral particle. We observed that the temperature of laser-treated samples did not increase by more than 2°C above room temperature as monitored by a thermocouple. MNV-1 is known to be highly thermostable, and readily tolerant of temperatures in excess of 60°C ,19. TherHowever, we note that it is possible that laser treatment could create “micro-thermal” effects where the laser energy might be absorbed by the viral particle itself and spread throughout the volume of the virus, heating up the viral particle and leading to the inactivation. In this micro-thermal scenario, one would expect that for a given laser power density, viruses with different sizes would be heated to different temperatures.For icosahedral viruses, if we consider a simple model calculation in which the capsid of the virus is assumed to completely absorb the incident photons and the energy thermalization within the viral particle is significantly faster than that between the viral particle and its surrounding, the total deposited energy is expected to be proportional to the cross-sectional area of the virus. In the micro-heating scenario, this deposited energy is to spread/thermalize over the whole volume of virus and increase the temperature of the virus.The total laser energy deposited is given byI is the laser intensity.where S is the cross-sectional area of the virus; m is the mass of the virus; c is the specific heat of the virus; ∆T is the increase of temperature of the virus; ρ is the density of the virus; V is the volume of the virus.I, if ρ,c are assumed to be constant, because S ∝ r2 and V ∝ r3, we haveFor a given laser intensity r is the radius of the virus.where I, the expected temperature increase (∆T) would be less for a larger virus than for a smaller virus.Therefore, for a given laser intensity 10 scale for MNV-1, HPV-16 (which is not shown here), respectively. The diameter for MNV-1 and the HPV-16 pseudovirion is approximately 30 and 55 nm, respectively. Thus, the observation that the larger icosahedral virus is more readily inactivated, argues against a micro-thermal mechanism. We note that even though the energy transfer between the virion and its environment is taken into account, because a larger virus has a larger surface area, resulting in greater energy loss, and thereby a lesser temperature increase (∆T), which again is in contradiction with our experimental observation of load reductions for MNV-1 and HPV-16.When we compare load reduction of MNV-1 and HPV -16 experiments under the same laser parameters, we find that the load reduction of 3.1 ± 0.1and 5.1 ± 0.5 in logFor this one-photon absorption scenario in which chromophores present in the sample solution, upon absorption of laser wavelength at 425 nm, may produce reactive oxygen species, leading to the inactivation of viral particles. This possibility is unlikely for the following reasons: (1) We have not observed any detectable absorption by MNV-1 viral particles at a wavelength of 425 nm; (2) MNV-1 used in this work were propagated in indicator-free media and substantially purified by sucrose gradient and Optiprep gradient centrifugation respectively, which presumably removed extraneous cellular proteins which might absorb blue laser light; (3) we have tested and found that the load reduction for the purified MNV-1 samples was consistent with that for which MNV-1 samples were not purified; (4) we tested the inactivation of MNV-1 by the USP laser with the orange juice as well as apple juice spiked with MNV-1. Within the experimental uncertainty, the load reduction of MNV-1 in MNV-1-spiked orange/apple juice was found to be the same as that in the buffer solution. Because both orange juice and apple juice are loaded with antioxidant – vitamin C, which is able to efficiently scavenges reactive oxygen species, these experimental results further confirm that the inactivation of MNV-1 by the USP laser cannot be due to the production of reactive oxygen species through the one-photon absorption process.ω0 is the angular frequency of the excited vibrational motion and ∆ωL is the full-width-at-half-maximum (FWHM) of the laser spectral width. Because the excited amplitude of vibration depends on the factor – -1[-1 for the inactivation of MNV-1 by a USP laser. This prediction is indeed observed in our experiments as described below.ISRS has been shown to be able to excite vibrational oscillations in solids and molecules ,20-26. Iut 2 cm-1, the ISRω0 to be 2.0 cm-1 for a non-enveloped, icosahedral virus with a diameter of 30 nm (28), × 10-6, 1.8 × 10-2, 3.7 × 10-1, 7.7 × 10-1, 8.9 × 10-1, 9.4 × 10-1 for ∆ωL = 0.25, 0.50, 1.0, 2.0, 3.0, 4.0 cm-1, respectively. In other words, the ISRS process predicts that in order to excite a sizable vibrational amplitude to break hydrogen bonds/hydrophobic contacts and achieve inactivation, the laser spectral width has to be comparable to, or larger than, the angular frequency of the oscillations. This prediction is indeed in consistence with the experimental results of Figure ωL is ≤ 0.5 cm-1; while significant inactivation is found for ∆ωL ≥ 2 cm- 1. Consequently, we attribute the inactivation mechanism of the non-enveloped, icosahedral MNV-1 by the USP laser to the ISRS process.To verify this prediction by ISRS inactivation mechanism, we have performed the inactivation experiments for MNV-1 as a function of the laser spectral width, which is shown in Figure 2) and the second part consisted of data within the observed sharp-drop of the fraction of survival (power density ≥ 80 MW/cm2). We have found that the first part of the data can be fit pretty well by the following function [To explain the data for the USP laser inactivation of MNV-1 in Figure function :y is the fraction of survival; x is the laser power density and A is the effective inactivation rate constant. Figure A = (0.017 ± 0.001)cm2/W.where 2. However, the qualitative behavior – sharp drop of the fraction of survival in this range of laser power densities, indicated that the effective inactivation rate in this range of laser power densities is much larger than that obtained from the first part of the data with laser power density up to 75 MW/cm2.We did not attempt to fit the second part of the data with laser power density ≥ 80 MW/cm2 and the other for laser power densities of 80 MW/cm2 and above. We note that the energy of a typical covalent bond in a biological system is about 4 eV or larger. In our laser experiments, the relatively low photon energy of 2.92 eV generated from the USP laser employed at a wavelength of 425 nm means that weaker hydrogen bonds or hydrophobic contacts can be broken but not the stronger covalent/ionic bonds. Furthermore, the broken hydrogen bonds and hydrophobic contacts in a molecule have been demonstrated to reform on a time scale of about 10 picoseconds at room temperature . With this knowledge in mind, we are able to explain the relatively small effective inactivation rate constant A (from equation (3)) deduced for laser power density of ≤ 75 MW/cm2. Because the laser power density is relatively small, the number of broken hydrogen bonds/hydrophobic contacts is relatively small; and since these broken bonds are rapidly reformed [2 simply reflects the threshold laser power density in which the number of broken bonds that can be easily reformed and the structure of virus can be easily restored for MNV-1. On the other hand, at the laser power density of ≥ 80 MW/cm2, inactivation for MNV-1 was dramatically enhanced, as indicated by the sharp-drop of the fraction of survival in this range of laser power density. We interpret this to be reminiscence of a deposited laser energy threshold through the ISRS process that once exceeded, results in so many simultaneously broken hydrogen bonds and hydrophobic contacts that the entire capsid is disintegrated spatially (as shown in TEM image of Figure 2, the capsid of MNV-1 begins to disassemble and the effective inactivation rate increases dramatically. It is worthwhile mentioning that the TEM images shown in Figure Considering the energy required to break hydrophobic contacts and hydrogen bonds, it is possible to explain the observed laser power density results shown in Figures perature , 31. WitWe note that in the previous studies of inactivation of enveloped virus like MCMV , the gloWe have investigated the inactivation mechanism of non-enveloped, icosahedral viruses - MNV-1 by USP laser irradiation. Possible mechanisms of inactivation are thoroughly examined. Real-time PCR measurements indicate that, within the amplicon size of 273 bp tested, USP laser irradiation does not degrade the genome of MNV-1. We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the TEM images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this efficient, non-invasive approach for the eradication of non-enveloped, icosahedral viruses, when employed in a continuous, syringe-pumped configuration, can be applied to the disinfection of pathogens in the pharmaceutical processes and disinfection of blood components for blood transfusion.The authors declare that they have no competing interests.SWDT plans and carries out the laser experiments, performs the PCR experiments, explains the experimental results, writes the manuscript; DHK prepares the virus samples, performs the PCR experiments, carries out the assays, helps explain the experimental results and writes the manuscript. CP carries out the laser experiments. SA plans the laser experiments, explains the experimental results. DSS performs the TEM experiments. TCW prepares the virus samples, carries out the assays. KTT plans and carries out the laser experiments, explains the experimental results and writes the manuscript. All authors read and approved the final manuscript.


To the Editor: Chikungunya virus (CHIKV) is endemic in rural tropical Africa and is penetrating urban areas in Asia. CHIKV is maintained in a sylvatic cycle that involves mosquitoes of the genus Aedes, primates, and rodents. CHIKV infection induces fever, arthralgia, and maculopapular rash. Hemorrhagic complications have been reported in some outbreaks, but a more specific symptom is severe arthralgia, often persistent, which results in long-lasting disability.After numerous cases of CHIKV infection had been reported in Comoros and Mauritius  collected on the island.Many samples, collected from patients during the outbreak, were sent to our laboratory  to identify the etiologic agent. Serum samples incubated with C6/36 cells according to previously published methods ; this finding could be relevant to the different role of these mosquito species in virus epidemiology.CHIKV has been isolated from To our knowledge, CHIKV has never been isolated from human corneas. In our study, the cornea sample was obtained from an asymptomatic donor whose serum contained immunoglobulin M (IgM) but not IgG to CHIKV; this finding suggests the patient was recently infected with CHIKV. The presence of CHIKV in corneal cells will have to be confirmed because the samples we studied also included sclera, vascular tissue that could contain circulating virions; however, no virus was detected in the patient's blood sample by CHIKV-specific RT-PCR assay (Our results indicate that CHIKV strains responsible for the outbreak in Reunion have a common origin and do not differ from strains circulating in East and Central Africa. More complete characterization of the 5 strains we report here, sequencing of the full-length genome, and phenotypic characterization of other CHIKV isolated in the area during the same period is currently underway in our laboratory.


The P gene of paramyxoviruses is unique in producing not only P but also “accessory” C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C’, C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported. Paramyxoviridae of the order Mononegavirales including some of the most important and ubiquitous disease-causing viruses of humans and animals, such as parainfluenza viruses, measles virus, mumps virus, Hendra virus, Nipah virus, human metapneumovirus, Newcastle disease virus, canine distemper virus, and rinderpest virus. SeV contains a nonsegmented, negative-strand RNA genome of 15,384 nucleotides (nt) encoding six viral structural proteins, a nucleoprotein (N), a highly phosphorylated component of the viral RNA-dependent RNA polymerase (vRdRp) complex (P), a matrix protein (M), a glycoprotein with membrane fusion activity (F) and hemagglutinin-neuraminidase activity (HN), and a large catalytic subunit of the vRdRp complex (L), tandemly in this order Sendai virus  is a prototype of the family Since C protein was first found in SeV-infected cells but apparently absent in virions, they were termed nonstructural proteins With the development of reverse genetics systems, which allow the recovery of infectious, negative-sense viruses from full-length cDNA of viral genomes, various recombinant viruses, possessing desired mutations or lacking viral proteins have been generated. A SeV recombinant 4C(-) lacking expression of all four C proteins was successfully generated using this type of system by introducing three stop codons onto upstream of the C ORF without alteration of the P polypeptide . OverallA field pathogenic strain Ohita was rendered less pathogenic for mice through adaptation to growth in LLC-MK2 cells As an “accessory” protein, the antagonism of C protein against host innate immunity is not essential for virus replication itself, regardless of its importance to the pathogenesis in small animals. However, later studies revealed that SeV C protein also exerts other important functions at multiple stages of virus replication, such as inhibition of virus-induced apoptosis In this paper, we examined the effect of serial passages of the 4C(-) SeV through embryonated chicken eggs, and found a quick emergence of revertant viruses caused by markedly rapid and high levels of accumulation of U-to-C transition mutations to recover C protein expression, suggesting that the lack of C protein lead unexpectedly to strong selective pressures on virus replication, and supporting the above-mentioned importance of C protein in the viral life cycle.LLC-MK2 cells were maintained in Dulbecco’s minimum essential medium  supplemented with 5% fetal calf serum  and penicillin-streptomycin (Invitrogen) at 37°C. The Polyclonal antibody (pAb) against whole virions of SeV was described previously 4 CIU/ml), kindly provided by A. Kato, which was allantoic fluid harvested from eggs after two successive passages of cells of the initial viral rescue experiment from the 4C(-) cDNA  virus using the P2 stock (7.2 × 10(-) cDNA . Approxi passage . For theLLC-MK2 cells cultured in 35 mm-diameter dishes were infected with the indicated viral fluid stocks at a multiplicity of infection (MOI) of 5. After a 1 h-incubation at 37°C, inocula were removed, and cells were washed with phosphate-buffered saline (PBS) three times, and incubated with serum-free MEM at 37°C. The culture medium was harvested at the indicated time points, and titrated as described above.ACCAAACAAGAGAAAAAACATGTATGGG) +3SeVZ1066 (AGGATACAGATAAAGGGAGC), 5SeVZ995 (TATTAATAAGCTTAGAAGCC).Viral RNA was prepared from each allantoic fluid stock using a HighPure Viral Nucleic Acid kit (Roche Diagnostics) and subjected to RT-PCR using a One-step RT-PCR Kit (QIAGEN) with the primer sets of 5SeVZ1 (GCGGTAAGTGTAGCCGAAGCCG), 5SeVZ1508 (GGCTGAACGGTTAGAGGAGG)+rev743NE4R (AGGACCCCAG TTACTCTTGC), and 5SeVZ2449 (GAAGCATGGAGCCTGGCAGC) +3SeVZ3572 (GGTAGGATGCCTCACCCGGG). For direct nucleotide sequencing, the RT-PCR products were sequenced twice in forward and reverse directions using a 3100 Genetic Analyzer (Applied Biosystems) with the same primer pairs used for the RT-PCR. The RT-PCR products prepared above were also subcloned into the pUC18 plasmid, and the resultant plasmid clones were subjected to DNA sequencing. At least twenty plasmid clones for each virus stock were analyzed.+3SeVZ1843 -P8 have been deposited in the GenBank database under accession numbers AB753441-9.LLC-MK2 cells cultured in 35mm-diameter dishes were infected with each of the 4C(-) fluid stock at an MOI of 1. After 1h at 37°C, inocula were removed, and cells were washed with PBS three times and incubated with serum-free MEM at 37°C. At 24 h post-infection (p.i.), cells were suspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer  and analyzed by SDS-PAGE (15%), followed by Western blotting with anti-SeV P or anti-SeV C pAb.The 4C(-) recombinant virus was generated by introducing several amino acid substitutions within the C proteins without changing of the P polypeptide . To sile8 and 8.3 × 107 CIU/ml, and 4 HAU higher than that of P2 were obtained, respectively  obtained by two consecutive egg passages of the 4C(-) transfectant was further passaged through embryonated chicken eggs twice in three individual experiments, and finally the allantoic fluid stocks S1-P4, S2-P4 and S3-P4 with titers of 4.1 × 10ectively . Unexpecectively .In terms of recovery of C protein expression of the 4C(-) virus, effects of further egg passages of two series of the P4 stocks, the C-recovered S2-P4 and the C-lacking S3-P4 up to the seventh or eighth passage, respectively, were examined . AllantoTo examine the effect of the recovery of C protein expression on virus replication, we compared one-step growth kinetics of the C-recovered S3-P8 stock with those of the C-lacking S3-P4 and the WT (WT-P1), which were propagated by the WT transfected cell lysate once in embryonated chicken eggs . ConsistTo identify alterations of nucleotides in the 4C(-) genomes causing the recovery of C protein expression, we prepared bulk RNA from allantoic fluid stocks of the S3 series of fourth to eighth passages, and their nucleotide sequences of a region covering from N to P genes were compared to that of the original 4C(-) genome  and 5. UAs mentioned above, bulk nucleotide sequencing of the viruses included in the S3-P8 stock revealed that markedly quick accumulation of U-to-C transition mutations occurred within a small 100-nt region of the 4C(-) genome after only seven or eight passages through eggs. This raises possibilities that a single type of revertant virus emerged at an earlier passage became selectively dominant during further passages, or that multiple types of mutated virus with better growth than the original 4C(-) virus emerged and dominated in the fluid stocks at higher passages.To examine these possibilities, the composition of viruses in the allantoic fluid stocks of S3-P6 to -P8 was analyzed in terms of nucleotide substitutions within the 1,801 - 2,100-nt region of their genomes . RT-PCR In the WT-P8 stock, there were a variety of viruses having mutations of U-to-C as well as other types, which were observed throughout the region randomly, and the occupancy rate of each mutation against the virus population of the fluid stock was less than 5% . In contThe rapid accumulation of U-to-C transitions in a limited region of the 4C(-) genome through egg passages implies that this region is the one allowing hypermutation or that the mutations introduced in the 4C(-) virus cause strong selective pressure for efficient virus growth. To examine these possibilities, WT as well as a C recombinant virus, F170S, and a V-knock-out virus, V(-), generated from cDNAs were passaged eight times through eggs, as the S3 series of 4C(-) was. The F170S virus possesses an F170S mutation within the C protein and the V(-) virus lacks V protein expression, and both of them have been reported to show attenuated phenotypes on pathogenesis in mice A number of U-to-C transition mutations were accumulated in the S2-P7 stock within the region where similar mutations were observed for the S3-P8 stock, and resulted in five and nine amino acid substitutions in P and C polypeptides, respectively . Surpriset al.For the purpose of elucidating functions of the C protein, the C’/C(-) and 4C(-) recombinant viruses were first generated by Kurotani By taking advantage of the 4C(-) virus, a variety of fundamental aspects of SeV replication and pathogenesis have been revealed. Indeed, the SeV C protein has been reported to counteract the host type-I IFN system in multiple ways In the present study, we applied the 4C(-) recombinant to serial passages through eggs, and found quick emergence of the C-recovered revertant even after the sixth passage . As passGenomes of RNA viruses are error-prone during replication, because of a lack of proofreading and repair systems in the process of viral RNA synthesis. Thus, synonymous and non-synonymous nucleotide substitutions accumulate easily in RNA viruses, relative to DNA viruses. Non-synonymous substitutions (Ka) are caused by various selection pressures, while synonymous substitutions (Ks) are neutral, free from selection pressures, and accumulate as the rounds of replication increase. The ratio of Ka to Ks (Ka/Ks) represents whether an ORF has been under strong or weak selection pressures and tolerate the accumulation of mutations. Previously, we compared entire genome sequences of two SeV representatives, Hamamatsu and Z, each of distinct lineages, and calculated the Ka/Ks The mechanism by which the nucleotide substitutions accumulated quickly in a limited region of the 4C(-) genome has not been elucidated yet. However, incidental U-to-C transitions as well as other types of mutations were found randomly in the 4C(-) genome as observed in the WT, and a single type of the 4C(-) revertant became dominant in the population of the fluid stocks after several egg passages  and 7. TSuch biased transition hypermutation as observed for the 4C(-) SeV has been well characterized for measles virus (MeV). A host double-stranded RNA-specific adenosine deaminase ADAR1 is induced by MeV infection, and U-to-C transition substitutions are introduced exclusively within the M gene, resulting in emergence of defective viruses, which cause subacute sclerosing panencephalitis (SSPE) Altogether, the 4C(-) virus might be more deficient in replication than believed, because it seems to be exposed to severer selective pressures than expected, which might be why three or more blind passages through eggs were required for obtaining detectable titers of this virus. This suggests the critical importance of C protein in SeV replication, and that even in the experiments using the “C-lacking” 4C(-) virus performed previously: unidentified mutations in the genomes during egg passages might somewhat complement the functions, and obfuscate the “real” importance of C protein. Detailed characterization of the 4C(-) clones isolated from the fluid stocks would be interesting and important, and give a deep understanding of functions of C protein as well as paramyxovirus replication.