Viral Reduction Research Articles

Biological Evaluation of Uridine Derivatives of 2-Deoxy Sugars as Potential Antiviral Compounds against Influenza A Virus.

Influenza virus infection is a major cause of morbidity and mortality worldwide. Due to the limited ability of currently available treatments, there is an urgent need for new anti-influenza drugs with broad spectrum protection. We have previously shown that two 2-deoxy sugar derivatives of uridine (designated IW3 and IW7) targeting the glycan processing steps during maturation of viral glycoproteins show good anti-influenza virus activity and may be a promising alternative approach for the development of new anti-influenza therapy. In this study, a number of IW3 and IW7 analogues with different structural modifications in 2-deoxy sugar or uridine parts were synthesized and evaluated for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay in vitro, we showed that compounds 2, 3, and 4 exerted the most inhibitory effect on influenza virus A/ostrich/Denmark/725/96 (H5N2) infection in Madin-Darby canine kidney (MDCK) cells, with 50% inhibitory concentrations (IC50) for virus growth ranging from 82 to 100 (μM) without significant toxicity for the cells. The most active compound (2) showed activity of 82 μM with a selectivity index value of 5.27 against type A (H5N2) virus. Additionally, compound 2 reduced the formation of HA glycoprotein in a dose-dependent manner. Moreover, an analysis of physicochemical properties of studied compounds demonstrated a significant linear correlation between lipophilicity and antiviral activity. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities.

Interactive regulations by viruses and dissolved organic matter on the bacterial community

AbstractViruses and dissolved organic matter (DOM) are major top–down and bottom–up factors, respectively, which control the abundance and diversity of bacterial communities. However, the relative strength and interaction between these two regulators in different marine ecosystems are not fully understood. We applied viral reduction and cross‐transplantation of eutrophic estuarine and pristine coastal waters to examine the separate and combined effects of viruses and DOM on the bacterial community composition. We showed that bacteria were more abundant in the virus‐depleted groups, particularly in those from the eutrophic estuarine water (i.e., > 7 times higher, when compared with the virus‐rich groups). Cluster analyses showed that the bacterial communities in the virus‐rich groups were most similar to the in situ populations. Bacterial community richness, evenness and diversity were reduced at high DOM concentrations but were sustained by viruses. This allowed bacteria that were less vulnerable to viral infection to thrive (such as those in the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae, and SAR11), while suppressing those capable of fast growth at elevated DOM concentrations but which were more vulnerable to viral attack (such as those from the family Vibrionaceae). Our results showed that viral regulation occurring at the strain level can affect the bacterial community structure at higher phylogenetic levels. In addition, some less abundant groups, such as Vibrio and Alteromonas, might actually win the competition for resources, providing viral control is removed. Our results also showed that the population dynamics of certain bacterial groups was controlled by viruses, and the strength of such control was modulated by the concentration of DOM. We suggest that the composition of the bacterial community in aquatic environments results from the interactive regulation by DOM and viruses among other regulators.

Reduction of Zika virus infectivity in platelet concentrates after treatment with ultraviolet C light and in plasma after treatment with methylene blue and visible light.

Zika virus (ZIKV) has emerged as a potential threat to transfusion safety worldwide. Pathogen inactivation is one approach to manage this risk. In this study, the efficacy of the THERAFLEX UV-Platelets system and THERAFLEX MB-Plasma system to inactivate ZIKV in platelet concentrates (PCs) and plasma was investigated. PCs spiked with ZIKV were treated with the THERAFLEX UV-Platelets system at 0.05, 0.10, 0.15, and 0.20 J/cm2 UVC. Plasma spiked with ZIKV was treated with the THERAFLEX MB-Plasma system at 20, 40, 60, and 120 J/cm2 light at 630 nm with at least 0.8 µmol/L methylene blue (MB). Samples were taken before the first and after each illumination dose and tested for residual virus. For each system the level of viral reduction was determined. Treatment of PCs with THERAFLEX UV-Platelets system resulted in a mean of 5 log reduction in ZIKV infectivity at the standard UVC dose (0.20 J/cm2 ), with dose dependency observed with increasing UVC dose. For plasma treated with MB and visible light, ZIKV infectivity was reduced by a mean of at least 5.68 log, with residual viral infectivity reaching the detection limit of the assay at 40 J/cm2 (one-third the standard dose). Our study demonstrates that the THERAFLEX UV-Platelets system and THERAFLEX MB-Plasma system can reduce ZIKV infectivity in PCs and pooled plasma to the detection limit of the assays used. These findings suggest both systems have the capacity to be an effective option to manage potential ZIKV transfusion transmission risk.

English

Newcastle disease virus is a paramyxovirus which causes Newcastle disease in birds. Investigation was done on the effect of leaf extract of Phyllanthus amarus against Newcastle disease virus (NDV) using an in-ovo assay. Nine to eleven day-old viable embryonated chicken eggs (ECE) were used for the assay, these were divided into six groups of six eggs each. Methanol, aqueous and n-hexane extracts of the plant leaves were administered to the various groups at concentrations varying from 50 to 5 mg/ml. Embryonated eggs were incubated and embryo survival was monitored daily. Negative control and diluents control groups received phosphate buffer saline (PBS) and dimethly suphoxide (DMSO), respectively. The other group was uninoculated while a virus control group received 100 EID50/0.1 ml NDV alone. Bacteria free allantoic fluid from the embryonated eggs in different treatment groups were harvested and collected for spot hemagglutination (HA) test and HA assay to detect the presence of NDV viral particles and the viral titre, respectively. Leaf extracts were assayed for presence of phytochemicals and antioxidant potentials. It was observed from the results that the extract was toxic to the embryo at a concentration above 50 mg/ml and further results showed that the HA viral titre reduction was directly proportional to increasing extract concentration. The phytochemical assays of leaf extract revealed the presence of phytochemicals including alkaloids, tannins, saponins, flavonoids, phenols, steroids, glycosides. The current findings have demonstrated that leaf extract from P. amarus has potentials of medicinal value as well as antiviral activity against NDV in-ovo. Further experimental assays using live animal models are recommended to validate the use of P. amarus plant extract in therapeutic measure in chickens. Key words: Antiviral, Phyllanthus amarus, Newcastle disease virus (NDV), embryonated chicken eggs (ECE).

Invitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype.

To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7cells. Stable Huh-7cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000μg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7cells was determined by real-time PCR. RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50nmol/L), siRNA had no cytotoxic effects on Huh-7cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7cell clones at 24 (80%, P=0.013) and 48h (75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P<0.05) decrease in viral copy number. siRNA synergism (NS3-is33+NS3-is44) decreased viral load by 84% (P<0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P<0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88+NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P<0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P<0.05). siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7cells. Stable Huh-7cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.

SELECTION OF AN ANTIRETROVIRAL REGIMEN BASED ON THE RESISTANCE DATA

As part of the global strategy against HIV, UNAIDS formulated the 90-90--90 targets. The targets mean that 90% of people living with HIV and receiving treatment should have achieved viral suppression. One of the main obstacles to achieving the goal is HIV resistance to antiretroviral therapy. It occurs when the virus mutates and affinity of active ingredients of drugs for the corresponding viral proteins is reduced. Drugs differ by the genetic barrier. Non-nucleoside reverse-transcriptase inhibitors lose their ability to inhibit the replication after a single mutation, and ritonavir- boosted protease inhibitors - after 5--8th mutation. The key factor for adequate viral suppression and reduction of risks is good adherence to treatment. Medication non-adherence creates a favorable environment in the body for the evolution of the virus. In the Russian Federation, the prevalence of primary resistance reaches 6.02%, and poor adherence equals 26%. The data and the results of examinations for mutations should be considered when selecting an antiretroviral regimen and approach to patient to improve adherence.

Polysulfonate suramin inhibits Zika virus infection

Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3–5 log10 PFU viral reduction with IC50 value of ∼2.5–5 μg/ml (1.93 μM–3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1–24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.

Antiviral screen identifies EV71 inhibitors and reveals camptothecin-target, DNA topoisomerase 1 as a novel EV71 host factor

Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis.

Effect of (−)-epigallocatechin gallate at different pH conditions on enteric viruses

Epigallocatechin gallate (EGCG), a flavonoid from green tea, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. In this work, the antiviral activity of EGCG was assessed against hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, at different temperatures, contact times and pH conditions by cell-culture methods. EGCG was effective in reducing the titers of HAV and MNV in a dose-dependent manner at neutral pH and 25 and 37 °C, while no effect was reported at 4 °C. HAV and MNV infectivity was completely removed after overnight treatment with EGCG at 2.5 mg/mL at 37 °C. Furthermore, results also revealed that EGCG was very effective inactivating MNV and HAV at neutral and alkaline pH but was ineffective at pH 5.5. Results from cell-culture assays and viability RT-qPCR assays indicated that EGCG did not dramatically affect viral capsid, which instead may suffer subtle alterations of proteins. Moreover, HPLC/MS analysis of catechin solutions at different pHs indicated that antiviral activity was most likely due to catechin derivatives rather than EGCG itself, given the evolution of these compounds at the various pH conditions tested. These findings suggest that green tea catechins appear to be a suitable natural option for foodborne viral reduction.

Drug resistance-related mutations T369V/I in the connection subdomain of HIV-1 reverse transcriptase severely impair viral fitness

Fitness is a key parameter in the measurement of transmission capacity of individual drug-resistant HIV. Drug-resistance related mutations (DRMs) T369V/I and A371V in the connection subdomain (CN) of reverse transcriptase (RT) occur at higher frequencies in the individuals experiencing antiretroviral therapy failure. Here, we evaluated the effects of T369V/I and A371V on viral fitness, in the presence or in the absence of thymidine analogue resistance-associated mutations (TAMs) and assessed the effect of potential RT structure-related mechanism on change in viral fitness. Mutations T369V/I, A371V, alone or in combination with TAMs were introduced into a modified HIV-1 infectious clone AT1 by site-directed mutagenesis. Then, experiments on mutant and wild-type virus AT2 were performed separately using a growth-competition assay, and then the relative fitness was calculated. Structural analysis of RT was conducted using Pymol software. Results showed that T369V/I severely impaired the relative virus fitness, and A371V compensated for the viral fitness reduction caused by TAMs. Structural modeling of RT suggests that T369V/I substitutions disrupt powerful hydrogen bonds formed by T369 and V365 in p51 and p66. This study indicates that the secondary DRMs within CN might efficiently damage viral fitness, and provides valuable information for clinical surveillance and prevention of HIV-1 strains carrying these DRMs.

Comparative Inactivation of Murine Norovirus and MS2 Bacteriophage by Peracetic Acid and Monochloramine in Municipal Secondary Wastewater Effluent.

Chlorination has long been used for disinfection of municipal wastewater (MWW) effluent while the use peracetic acid (PAA) has been proposed more recently in the United States. Previous work has demonstrated the bactericidal effectiveness of PAA and monochloramine in wastewater, but limited information is available for viruses, especially ones of mammalian origin (e.g., norovirus). Therefore, a comparative assessment was performed of the virucidal efficacy of PAA and monochloramine against murine norovirus (MNV) and MS2 bacteriophage in secondary effluent MWW and phosphate buffer (PB). A suite of inactivation kinetic models was fit to the viral inactivation data. Predicted concentration-time (CT) values for 1-log10 MS2 reduction by PAA and monochloramine in MWW were 1254 and 1228 mg-min/L, respectively. The 1-, 2-, and 3-log10 model predicted CT values for MNV viral reduction in MWW were 32, 47, and 69 mg-min/L for PAA and 6, 13, and 28 mg-min/L for monochloramine, respectively. Wastewater treatment plant disinfection practices informed by MS2 inactivation data will likely be protective for public health but may overestimate CT values for reduction of MNV. Additionally, equivalent CT values in PB resulted in greater viral reduction which indicate that viral inactivation data in laboratory grade water may not be generalizable to MWW applications.

Antiviral Efficacy and Host Immune Response Induction during Sequential Treatment with SB 9200 Followed by Entecavir in Woodchucks.

SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-β, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.

Evaluation of Temporal Virological Responses to Interferon-α-2b plus Ribavirin among Genotype 3a Hepatitis C Virus-Infected Patients

Objectives: The present study aimed to examine the impact of rapid virological response (RVR) and early virological response (EVR) on sustained virological response (SVR) in chronic hepatitis C genotype 3a individuals. Methods: The patients were given antiviral therapy with IFN-α-2b, 3 million units 3 times a week and 800-1,200 mg of ribavirin daily adjusted to the patient's body weight (<60 kg 800 mg day−¹, and >60 kg 1,200 day−¹). The patients received this combination therapy for 24 weeks. The patients were evaluated for their viral load at week 4, 12, and 24 using RT-PCR. Results: Out of 1,471 patients, 43.3% showed a negative viral load in week 4, demonstrating RVR, whereas 56.6% maintained a high viral load. These were further separated based on viral reduction in their plasma: either negative for HCV-RNA at week 12 (n = 575), manifesting EVR, or showing a 2-log reduction in HCV viral load classified as partial EVR (PEVR; n = 259). The PEVR response was less (29.7%) compared with RVR (85.9%) and EVR (69.0%), although nonresponders were found in both groups. Conclusions: Individuals incompliant with their treatment who have a higher RVR significantly influence their SVR towards a better remission that can be treated within a short duration with standard treatment.

Inactivation efficiency and mechanism of UV-TiO2 photocatalysis against murine norovirus using a solidified agar matrix

Human norovirus (HuNoV) is the primary cause of viral gastroenteritis worldwide. Fresh blueberries are among high risk foods associated with norovirus related outbreaks. Therefore, it is important to assess intervention strategies to reduce the risk of foodborne illness. The disinfection efficiency of decontamination methods is difficult to evaluate for fruits and vegetables due to an inconsistent degree of contamination and irregular surface characteristics. The inactivation efficiency and mechanism of murine norovirus 1 (MNV-1, a surrogate for HuNoV) was studied on an experimentally prepared solidified agar matrix (SAM) to simulate blueberries using different wavelengths (A, B, C) of UV light both with and without TiO2 photocatalysis (TP). MNV-1 was inoculated on exterior and interior of SAM and inactivation efficiencies of different treatments were investigated using a number of assays. Initial inoculum levels of MNV-1 on the SAM surface and interior were 5.2logPFU/mL. UVC with TiO2 (UVC-TP) achieved the highest level of viral reduction for both externally inoculated and internalized MNV-1. Externally inoculated MNV-1 was reduced to non-detectable levels after UVC-TP treatment for 5min while there was still a 0.9 log viral titer after UVC alone. For internalized MNV-1, 3.2 log and 2.7 log reductions were obtained with UVC-TP and UVC alone treatments for 10min, respectively. The Weibull model was applied to describe the inactivation behavior of MNV-1, and the model showed a good fit to the data. An excellent correlation between the steady-state concentration of OH radicals ([OH]ss) and viral inactivation was quantified using a para–chlorobenzoic acid (pCBA) probe compound, suggesting that OH radicals produced in the UV-TP reaction were the major species for MNV-1 inactivation. Transmission electron microscopy images showed that the structure of viral particles was completely disrupted with UVC-TP and UVC alone. SDS-PAGE analysis showed that the major capsid protein VP1 was degraded after UVC-TP and UVC alone. Real-time RT-qPCR analysis showed that UVC-TP and UVC alone caused a reduction in the level of viral genomic RNA. Propidium monoazide (PMA) pretreatment RT-qPCR analysis showed that UVC-TP caused damage to the viral capsid protein in addition to viral genomic RNA. UVC both with and without TiO2 was more effective for MNV-1 inactivation than UVB and UVA. Thus, UVC-TP disinfection aimed to reduce levels of food-borne viruses can inactivate viruses present on the surface and internalized in the interior of blueberries.

Is hand hygiene frequency associated with the onset of outbreaks in pediatric long-term care?

Studies in adult long-term care facilities (LTCFs) have shown a correlation between hand hygiene (HH) and viral outbreak reduction, but no such studies have been conducted in pediatric LTCFs where the epidemiology of viral pathogens is different. We compared electronically monitored facility-wide HH frequency in the weeks immediately prior to outbreaks of acute respiratory or gastrointestinal infections versus control weeks in a 137-bed pediatric LTCF from October 2012-August 2015. Control weeks were the 8-14 day (control 1) and 15-21 day (control 2) periods prior to the onset of each outbreak. There was no difference in HH frequency in the weeks leading up to the outbreaks versus control weeks (odds ratio [OR], 1.0; 95% confidence interval CI, 1.00-1.001 using control 1 and OR, 1.0; 95% CI, 1.00-1.001 using control 2). Our findings differed from those in adult LTFCs, possibly because of the greater contact between residents and staff in the pediatric setting, increased susceptibility to viral pathogens because of immunologic immaturity, or differences in the types of pathogens prevalent in each setting. Although HH may be important for limiting the number of residents infected during outbreaks, we found no association between HH frequency and subsequent outbreak onset.

QMRAcatch: Human-Associated Fecal Pollution and Infection Risk Modeling for a River/Floodplain Environment.

Protection of drinking water resources requires addressing all relevant fecal pollution sources in the considered catchment. A freely available simulation tool, QMRAcatch, was recently developed to simulate concentrations of fecal indicators, a genetic microbial source tracking (MST) marker, and intestinal pathogens in water resources and to conduct a quantitative microbial risk assessment (QMRA). At the same time, QMRAcatch was successfully applied to a region of the Danube River in Austria, focusing on municipal wastewater emissions. Herein, we describe extension of its application to a Danube River floodplain, keeping the focus on fecal sources of human origin. QMRAcatch was calibrated to match measured human-associated MST marker concentrations for a dry year and a wet year. Appropriate performance characteristics of the human-associated MST assay were proven by simulating correct and false-positive marker concentrations, as determined in human and animal feces. With the calibrated tool, simulated and measured enterovirus concentrations in the rivers were compared. Finally, the calibrated tool allowed demonstrating that 4.5 log enterovirus and 6.6 log norovirus reductions must be achieved to convert current surface water to safe drinking water that complies with a health-based target of 10 infections person yr. Simulations of the low- and high-pollution scenarios showed that the required viral reductions ranged from 0 to 8 log. This study has implications for water managers with interests in assessing robust catchment protection measures and water treatment criteria by considering the fate of fecal pollution from its sources to the point of abstraction.

Current status and new developments in the production of plasma derivatives

Plasma fractionation is a unique biotechnology and bioengineering industry characterized by its capacity to extract a diverse range of protein therapeutics that are often the only options for treating life‐threatening pathological conditions of patients suffering from bleeding, immunological disorders, infections or trauma. Current plasma products include several coagulation factors, polyvalent and hyperimmune immunoglobulins (Igs), albumin, protease inhibitors and anticoagulants. In many pathological situations, these products are used for long‐term replacement therapy to compensate for a congenital or acquired protein deficiency. Most products are obtained by plasma fractionation technologies that derive from the cold ethanol fractionation process developed in the mid‐/late 1940s by Cohn and collaborators. Still, in the last 20 years, this core technology has undergone substantial evolutions illustrated by the integration of several viral reduction steps for all products and by the introduction of chromatographic purification steps to extract labile proteins from cryoprecipitate‐poor plasma. The most recent technological changes, intended mostly to improve recoveries and remove trace protein contaminants, concern the production process of intravenous Igs through implementing chromatographic steps and caprylic acid treatment in early stages of the ethanol fractionation process. A number of new fractionation technologies are currently under development and have not been licensed at least for the time being by major regulatory authorities. One target of these technologies is to improve the recovery of the most important plasma protein products, under conditions facilitating large‐scale implementation in emerging countries.

Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis virus surrogate Langat virus.

BackgroundLongicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat virus (LGTV), a tick-borne flavivirus.MethodsLongicin P1 and P4 peptides were chemically synthesized. Antiviral activity of the longicin peptides against LGTV was evaluated through in vitro virucidal assays, wherein the antiviral efficacy was determined by reduction in number of viral foci and virus yield. Additionally, longicin P4 was also tested for its activity against human adenovirus, a non-enveloped virus. Lastly, to assess the importance of longicin on the innate antiviral immunity of H. longicornis ticks, gene silencing through RNAi was performed.ResultsLongicin P4 produced significant viral foci reduction and lower virus yield against LGTV, while longicin P1 failed to demonstrate the same results. Conversely, both longicin partial analogs (P1 and P4) did not show significant antiviral activity when tested on adenovirus. In addition, longicin-silenced ticks showed significantly higher virus titer after 7 days post-infection but a significantly lower titer was detected after an additional 14 days of observation as compared to the Luc dsRNA-injected ticks. Mortality in both groups did not show any significant difference.ConclusionOur results suggest that longicin P4 has in vitro antiviral activity against LGTV but not against a non-enveloped virus such as adenovirus. Likewise, though most cationic antimicrobial peptides like longicin act directly on target membranes, the exact mechanism of membrane targeting of longicin P4 in enveloped viruses, such as LGTV, requires further investigation. Lastly, while the in vitro virucidal capacity of longicin P4 was confirmed in this study, the role of the endogenous tick longicin in the antiviral defense of H. longicornis against LGTV still remains to be demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1344-5) contains supplementary material, which is available to authorized users.

Continuous viral inactivation at low pH value in antibody manufacturing

Due to the evolving biopharmaceutical market, small scale and flexible production processes for continuous antibody manufacturing become more important. Consequently, various continuously operated processing steps like perfusion fermentation or continuous chromatography have been developed. However, no continuously operated viral inactivation at low pH value is available so far. In this article, the coiled flow inverter (CFI) is identified as a suitable reactor for this application and two different design approaches are presented. The logarithmic reduction value (LRV) approach targets at the same LRV in the continuous viral inactivation when compared to current batch operation. The minimum residence time (MRT) approach guarantees a residence time for all molecules entering the continuous viral inactivation of at least the inactivation time of the batch inactivation step. The viral reduction value and the monomer loss during inactivation are characterized for each design approach. Moreover, a linear regression model is developed, which predicts the Bodenstein number as a function of two CFI design parameters under defined boundary conditions. This model allows designing the CFI according to the inactivation time and the chosen design approach.

Discovery of antiviral molecules for dengue: In silico search and biological evaluation

BackgroundDengue disease is a global disease that has no effective treatment. The dengue virus (DENV) NS2B/NS3 protease complex is a target for designing specific antivirals due to its importance in viral replication and its high degree of conservation. MethodsNS2B/NS3 protease complex structural information was employed to find small molecules that are capable of inhibiting the activity of the enzyme complex. This inhibitory activity was probed with in vitro assays using a fluorescent substrate and the complex NS2B/NS3 obtained by recombinant DNA techniques. HepG2 cells infected with dengue virus serotype 2 were used to test the activity against dengue virus replication. ResultsA total of 210,903 small molecules from PubChem were docked in silico to the NS2B/NS3 structure (PDB: 2FOM) to find molecules that were capable of inhibiting this protein complex. Five of the best 500 leading compounds, according to their affinity values (−11.6 and −13.5 kcal/mol), were purchased. The inhibitory protease activity on the recombinant protein and antiviral assays was tested. ConclusionsChemicals CID 54681617, CID 54692801 and CID 54715399 were strong inhibitors of NS2B/NS3, with IC50 values (μM) and percentages of viral titer reductions of 19.9, 79.9%; 17.5, 69.8%; and 9.1, 73.9%, respectively. Multivariate methods applied to the molecular descriptors showed two compounds that were structurally different from other DENV inhibitors. General significance: This discovery opens new possibilities for obtaining drug candidates against Dengue virus.