Viral Reduction Research Articles

Stability of human enteric viruses in seawater samples from mollusc depuration tanks coupled with ultraviolet irradiation

To evaluate the stability in seawater of human adenovirus (HAdV2), murine norovirus (MNV-1) and hepatitis A virus (HAV) in a shellfish depuration system with and without ultraviolet (UV) treatment. Seawater was seeded with viruses and disinfected using a 36 W lamp. Samples were collected at 24, 48, 72, 96 and 120 h; viruses were concentrated and the viral decay was evaluated using molecular and cell culture methods. Based on the molecular results, at 120 h of disinfection, there was a reduction of more than 3 log(10) for HAdV2 and HAV; MNV-1, a 4.5 log(10) reduction was observed at 72 h. Infectious MNV-1 was not detected after 72 h of treatment; while HAdV2 remained infectious. Seawater not treated demonstrated a progressive viral reduction for the three viruses tested. The UV reduced the number of viral particles, and the results indicate there is natural and gradual decrease of viral load and viability in seawater. UV irradiation is the method of choice for shellfish depuration in many countries; this work showed useful information about the viral stability in seawater and application of UV to water disinfection to be used in shellfish depuration tanks.

Ramsay Hunt syndrome with trochlear nerve involvement and EEG abnormalities: Multicranial neuritis or encephalitis?

Viral kinetics and host interleukin 28B (IL-28B) genotype determine treatment outcome in hepatitis C virus genotype 1 (HCV-1) infection.We aimed to explore the interplay between interferon responsiveness at treatment week 4 and IL28B genotype in the achievement of a sustained virological response (SVR; undetectable HCV RNA 24-weeks after end-of-treatment).Rs8099917 genotypes were determined in 528 HCV-1 patients with peginterferon/ribavirin. Interferon responsiveness were evaluated by the degree of week 4 viral reduction: <1 log10 IU/mL, 1–2 logs10 IU/mL, 2–3 logs10 IU/mL, 3–4 logs10 IU/mL and ≥4 logs10 IU/mL reduction and/or undetectable HCV RNA, respectively.The SVR rate was significantly higher in patients with great interferon responsiveness at week 4. A great interferon responsiveness was associated with younger age (P < 0.0001), lower body mass index (P = 0.0056), lower aspartate aminotransferase levels (P = 0.0009), higher hemogloblin concentration (P = 0.0033), higher platelet counts (P < 0.0001), male gender (P < 0.0001) and rs809997 TT-genotype (P < 0.0001). Comparing to non-TT genotype patients, TT genotype patients had a significantly higher SVR rate with moderate viral reduction (1–3 logs10 IU/mL) at week 4 (58.9% vs. 18.2%, P < 0.001), and the SVR rate did not differ between TT/non-TT patients on the extreme ends (<1 or >3 log10 IU/mL reduction) of week 4 interferon responsiveness. For non-TT genotype carriers who were with <3 logs10 reduction, none (0/15) could have a complete early virological response and only 10.9% (7/64) of the patients had an SVR.More profound interferon responsiveness is mandatory for HCV-1 patients with unfavorable IL-28B genotype.

Statistical evaluations of viral clearance studies for biological products

Since most biological products are derived from living cell culture, it is possible that viral contaminants be transmitted to the final product. Regulatory guidance requires that viral clearance studies be conducted to demonstrate the capacity of the production process in viral removal and inactivation. The key is accurate estimation of viral titer and reduction factor (RF), defined as the difference in log10 virus titers before and after each step of purification. Darling et al. (1998) [1] suggested a method for analysis of clearance studies. However it is unable to establish an estimate of RF when the post-process viral counts are zero. In this paper, we provide theoretical justification of the method based on normal distribution and discuss the caveats regarding the degrees of freedom. We propose two alternative methods under the assumption that the number of plaques follows a Poisson distribution. Through simulation studies, the Poisson-based methods are shown to provide better estimates of viral titers. Under the Poisson model, we also derive a method to calculate the exact confidence limits for the viral titer and reduction factor even if the post-process viral counts are zero. The use of the methods is illustrated through numerical examples.

Extended peginterferon plus ribavirin treatment for 72 weeks versus standard peginterferon plus ribavirin treatment for 48 weeks in chronic hepatitis C genotype 1 infected slow-responder adult patients.

The standard length of peginterferon plus ribavirin treatment for chronic hepatitis C virus (HCV) genotype 1 infected patients is 48 weeks. However, the number of patients demonstrating a sustained virological response is not high. In order to improve sustained virological response, extending the length of the treatment period has been suggested. To study the benefits and harms of extended 72-week treatment in comparison with 48-week treatment with peginterferon plus ribavirin in patients with chronic HCV genotype 1 infection who have shown a slow antiviral response. We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and LILACS until November 2011. We identified further trials by reviewing reference lists and contacting principal authors. Trials were eligible for this review if they included patients infected with hepatitis C virus genotype 1 who had a slow antiviral response, and if those patients were randomised to completing 72 weeks versus 48 weeks of treatment with pegylated interferon and ribavirin. Two authors independently assessed the trials for risk of bias, and extracted the data. The primary outcomes were overall mortality, liver-related mortality, and liver-related morbidity. We extracted data separately according to two definitions of slow responders: 1) patients with ≥ 2 log viral reduction but still detectable HCV RNA after 12 weeks of treatment and undetectable HCV RNA after 24 weeks of treatment; 2) patients with detectable HCV RNA after four weeks of treatment. We calculated risk ratios from individual trials as well as in the meta-analyses of trials. We included seven trials with 1369 participants. All trials had high risk of bias. Five trials used our first definition of slow responders, and three other trials (including one that used both definitions) used the second definition. None of the included trials mentioned our primary outcomes. However, regarding the secondary outcomes, extension of the treatment period to 72 weeks increased the sustained virological response according to both definitions (71/217 (32.7%) versus 52/194 (26.8%); risk ratio (RR) 1.43, 95% confidence interval (CI) 1.07 to 1.92, P = 0.02, I(2) = 8%; and 265/499 (53.1%) versus 207/496 (41.7%); RR 1.27, 95% CI 1.07 to 1.50, P = 0.006, I(2) = 38%), with a risk difference of 0.11 and calculated number needed to treat of nine. The end of treatment response was not significantly different between the two treatment groups. The number of participants who relapsed virologically was found to be lower in the groups that had been treated for 72 weeks using both definitions (27/84 (32.1%) versus 46/91 (50.5%); RR 0.59, 95% CI 0.40 to 0.86, P = 0.007, I(2) = 18%, 3 trials; and 85/350 (24.3%) versus 146/353 (41.4%); RR 0.59, 95% CI 0.47, 0.73, P < 0.000001, I(2) = 0%, 3 trials). The length of treatment did not significantly affect the adherence (247/279 (88.5%) versus 252/274 (92.0%); RR 0.95, 95% CI 0.84 to 1.07, P = 0.42, I(2) = 69%, 3 trials). In the single trial that reported adverse events, no significant difference was seen between the two treatment groups. This review demonstrates higher a proportion of sustained virological response after extension of treatment from 48 weeks to 72 weeks in HCV genotype 1 infected patients in whom HCV RNA was still detectable but decreased by ≥ 2 log after 12 weeks and became negative after 24 weeks of treatment, and in patients with detectable HCV RNA after four weeks of treatment with peginterferon plus ribavirin. The observed intervention effects can be caused by both systematic error (bias) and random errors (play of chance). There was no reporting on mortality and the reporting of clinical outcomes and adverse events was insufficient. More data are needed in order to recommend or reject the policy of extending the treatment period for slow responders.

Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

Factors Influencing Viral Clearing and Renal Function During Polyomavirus BK–Associated Nephropathy After Renal Transplantation

The course of BK virus nephropathy (BKVN) is difficult to predict. Between 2008 and 2010, we diagnosed BKVN in 46 (5.5%) of 859 patients with transplant biopsies by simian virus 40 (SV40) staining and routine serum polymerase chain reaction. We measured the influence of different variables on glomerular filtration rate (ΔGFR increasing or decreasing) and the time for viral polymerase chain reaction reduction by 1 log (≤13 or >13 weeks). At diagnosis, we either reduced calcineurin inhibitor (CNI) and mycophenolate mofetil by 30% to 50% (n=23), or we switched from CNI to mammalian target of rapamycin (mTOR) inhibitor (n=7) or from CNI to mTOR inhibitor as a second step in patients with protracted viral reduction (n=16). Results are the following: GFR stabilized or increased in 61% of patients and decreased in 39% (graft failure, 15%). Viral reduction by 1 log was rapid in 54% (≤13 weeks) and slow in 46% (>13 weeks). Rapid viral reduction was associated with stable or increasing GFR (84%), compared with slow viral reduction (33%; P=0.0004). High peak viral load, tacrolimus treatment, and late diagnosis (biopsy for cause vs. protocol biopsy) had a negative influence on GFR and viral reduction time. Defining 1-log viral load reduction as an event, tacrolimus compared with cyclosporine was associated with slow viral reduction (P=0.0043). In 88% of patients with slow viral reduction, the secondary switch from CNI to mTOR inhibitor favored viral load decrease. We conclude that peak viral load, tacrolimus treatment, delayed diagnosis, and viral reduction time influence outcomes in patients with BKVN.

Photocatalytic inactivation of influenza virus by titanium dioxide thin film

Titanium dioxide (TiO(2)) under ultraviolet (UV) light produces a strong oxidative effect and may therefore be used as a photocatalytic disinfectant. Although many studies on the photocatalytic inactivation of bacteria have been reported, few studies have addressed virus inactivation. In the present study, we demonstrated the inactivation of influenza virus through TiO(2) photocatalysis using TiO(2) nanoparticles immobilized on a glass plate. The influences of the UV intensity, UV irradiation time and bovine serum albumin (BSA) concentration in the viral suspensions on the inactivation kinetics were investigated. Additionally, we also determined whether the International Organization for Standardization (ISO) methodology for the evaluation of antibacterial activity of TiO(2) photocatalysis could be applied to the evaluation of antiviral activity. The viral titers were dramatically reduced by the photocatalytic reaction. Even with a low intensity of UV-A (0.01 mW cm(-2)), a viral reduction of approximately 4-log(10) was observed within a short irradiation time. The viral inactivation kinetics were associated with the exposure time, the UV intensity and the BSA concentration in virus suspensions. These results show that TiO(2) photocatalysis could be used to inactivate the influenza virus. Furthermore, a minor modification of the ISO test method for anti-bacterial effects of TiO(2) photocatalysis could be useful for the evaluation of antiviral activity.

Plasma fractionation

The first plasma fractionation process was developed in the 1940s by Cohn and co‐workers to prepare albumin and immunoglobulins. It relies on sequential precipitation steps at negative temperatures, different ethanol concentrations and various pH to segregate bulk plasma proteins. The ‘Cohn procedure’ has been modified over the years but largely remains the core fractionation process in use up to now. The introduction of cryoprecipitation (thawing of plasma at 2–4°C), as the first plasma fractionation step, to isolate a crude factor‐VIII fraction, and the integration of chromatographic steps to extract labile plasma or trace plasma proteins, have eventually shaped the modern fractionation technologies into complex procedures. Viral inactivation and removal treatments are performed to prevent the risks of transmission of enveloped and non‐enveloped viruses. Viral inactivation steps are applied most often on pre‐purified fractions, the downstream steps being used to remove protein contaminants and viral inactivation agents. Depending upon products and protein stability, treatments include incubations with combinations of solvent (TnBP) and detergent (e.g. Tween 80 or Triton X‐100) (S/D), pasteurization (heat‐treatment in the liquid state at 60°C for 10 h in the presence of stabilizers), as well as low pH or caprylic acid treatments (these last two treatments are restricted to immunoglobulins). Nanofiltration, to capture viruses on nm‐membranes, is typically performed on the final purified protein fractions prior to sterile filtration and aseptic filling. Terminal dry‐heat treatment at 80–100°C is used for some coagulation factor preparations. Modern plasma protein products have an unprecedented level of quality and safety when there is careful process control and monitoring together with sensitive test methods to detect potentially harmful contaminants, such as procoagulant impurities. Since the implementation of validated robust viral reduction treatments, plasma products have a high viral safety profile including against emerging agents (WNV, Dengue virus, Chikungunya virus). Experimental spiking studies suggest that several fractionation steps are capable of removing prions causing variant Creutzfeldt‐Jakob disease. Alternative fractionation processes based exclusively on chromatography have been developed at pilot scale and need further validations to demonstrate the quality, safety and consistency of resulting plasma products. A mini‐pool viral inactivation and fractionation process relying on single‐use equipment is also being developed to facilitate the access to safer plasma components, including plasma for transfusion, cryoprecipitate, prothrombin complex, immunoglobulins and fibrin sealant, in particular in developing countries.

Foreword

von Willebrand disease (VWD) is probably the most common congenital bleeding disorder while haemophilia, although relatively rare, remains the best characterized. Advances in the prevention and treatment of bleeding episodes in both conditions have translated into improved quality of life and survival across all age groups. However, improved survival does not come without its own side effect. As patients advance in age, several previously unrecognized morbidities continue to manifest. In this supplement, we highlight the most relevant complications in the ageing population and overview current approaches to their management, while realizing areas of unmet needs. The other focus of the supplement is to shed light upon persisting challenges in the prophylactic treatment of bleeding episodes in patients with haemophilia and VWD, within all age groups. Since the middle of the last century, clotting factor replacement for persons with haemophilia has evolved from plasma cryoprecipitate to plasma protein-free recombinant factor. Reactionary focus on pathogen transmission was addressed in the late 1980s with viral reduction measures for plasma-derived clotting factor (pd-CF) and the development of recombinant clotting factor (rCF) concentrates. What has resurfaced is the unsolved dilemma of inhibitor development in a substantial number of individuals with haemophilia A, and possibly a differential inhibitor induction rate among recipients of pd-CF containing von Willebrand factor (VWF) and rCF. In addition, renewed attention on the profile of the inhibitor patient has incriminated genetic predispositions, timing and intensity of exposure to CFs and temporally associated immunological danger signals. Cogent studies to address these questions, in the United States and internationally, include SIPPET, the CDC Inhibitor Study and the Haemophilia Inhibitor Genetics Study (HIGS). In addition, the means by which these inhibitors can be eradicated using ITI procedures are under investigation. Prophylactic treatment has been recommended for children with severe haemophilia in view of its efficacy in reducing the number of bleeds and therefore preventing the development of haemophilic arthropathy. Nonetheless, the ideal age for starting prophylaxis and the optimal dosing schedule, remain unclear. Moreover, the fact that primary prophylactic treatment with clotting factor has dramatically preserved the joints of younger persons with haemophilia brings into question whether the whole of this benefit continues throughout the adult life of haemophilia patients. It is not known whether intensive episodic or prolonged regular prophylaxis contributes to ageing-related disorders, such as ischaemic heart disease or renal disease, or prevents osteoporosis, in persons with haemophilia; a new focus for the community of haemophilia clinicians and scientists to address. Various investigator-initiated studies are underway in the US, keeping in mind that prospective collaborations are necessary to study this important subset of individuals with haemophilia. Plasma-derived concentrates containing both VWF and coagulation factor VIII (FVIII) have been available since the early 1980s for the treatment of severe VWD with proven haemostatic efficacy and safety. Several clinical studies are also ongoing to evaluate the efficacy and safety of novel recombinant products or single VWF replacement without FVIII, and the benefits of prophylactic use in this patient population. Nonetheless, establishing specific management guidelines, while utilizing the longstanding experience from patients with haemophilia, is warranted. A special focus on women affected with VWD is also called for, as they can experience significant gynaecological and obstetrical bleeding episodes that not only affect their physical health but also their psychosocial well-being. The author received an honorarium from Grifols S.A. for participating in the symposium and production of the article. The author thanks Content Ed Net for providing valuable editorial assistance in the preparation of the article; funding for this assistance was provided by Grifols S.A.

Leflunomide Efficacy and Pharmacodynamics for the Treatment of BK Viral Infection

BK virus is an infection in kidney transplantation patients jeopardizing graft survival. Unfortunately, there is no consensus on treatment of BK viremia and nephropathy. Leflunomide has been studied for the treatment of BK viremia and nephropathy, but there are limited data on the utility of leflunomide therapeutic drug monitoring. This study aimed to determine if a pharmacodynamic relationship exists between BK viral load reduction and leflunomide metabolite, A77 1726, serum concentrations. This study was a retrospective, single-center, longitudinal analysis of patients identified with BK viremia with or without nephropathy. Patients were grouped according to whether they received leflunomide. All BK viral PCR and A77 1726 concentrations were analyzed to determine pharmacodynamics, and were correlated with clinical outcomes. Of 76 patients identified, 52 received leflunomide therapy and 24 did not. Patients who received leflunomide were further analyzed according to A77 1726 concentrations and BK clearance; there was no difference in BK clearance. There was a lack of correlation between A77 1726 concentrations and log change in BK viral PCR concentration. Multivariate analysis demonstrated that mycophenolate mofetil discontinuation, BK viremia without nephropathy, and mean BK viral load were significantly associated with BK viral clearance; leflunomide use lacked this association. Pharmacodynamic analysis revealed no association between A77 1726 concentrations and BK viral PCR reductions. Multivariate analysis demonstrated that leflunomide therapy was not associated with BK viral clearance. Randomized studies are needed to determine the utility of leflunomide for BK viremia and nephropathy.

Expression of infectious pancreatic necrosis virus (IPNV) VP2–VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

Antiviral Ability ofKalanchoe gracilisLeaf Extract against Enterovirus 71 and Coxsackievirus A16

Pandemic infection or reemergence of Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) occurs in tropical and subtropical regions, being associated with hand-foot-and-mouth disease, herpangina, aseptic meningitis, brain stem encephalitis, pulmonary edema, and paralysis. However, effective therapeutic drugs against EV71 and CVA16 are rare. Kalanchoe gracilis (L.) DC is used for the treatment of injuries, pain, and inflammation. This study investigated antiviral effects of K. gracilis leaf extract on EV71 and CVA16 replications. HPLC analysis with a C-18 reverse phase column showed fingerprint profiles of K. gracilis leaf extract had 15 chromatographic peaks. UV/vis absorption spectra revealed peaks 5, 12, and 15 as ferulic acid, quercetin, and kaempferol, respectively. K. gracilis leaf extract showed little cytotoxicity, but exhibited concentration-dependent antiviral activities including cytopathic effect, plaque, and virus yield reductions. K. gracilis leaf extract was shown to be more potent in antiviral activity than ferulic acid, quercetin, and kaempferol, significantly inhibiting in vitro replication of EV71 (IC50 = 35.88 μg/mL) and CVA16 (IC50 = 42.91 μg/mL). Moreover, K. gracilis leaf extract is a safe antienteroviral agent with the inactivation of viral 2A protease and reduction of IL-6 and RANTES expressions.

Atividade antiviral de extratos de plantas medicinais disponíveis comercialmente frente aos herpesvírus suíno e bovino

O presente trabalho teve como objetivo pesquisar a atividade antiviral in vitro de plantas medicinais disponíveis comercialmente sobre herpesvírus suíno (SuHV-1) e bovino (BoHV-1). As espécies adquiridas foram Mikania glomerata, Cymbopogon citratus, Equisetum arvense, Peumus boldus, Solanum paniculatum, Malva sylvestris, Piper umbellatun e Solidago microglossa. A citotoxicidade dos extratos foi avaliada na linhagem celular MDBK pelas alterações morfológicas das células e obtenção da concentração máxima não citotóxica (CMNC) de cada planta. A atividade antiviral foi realizada com os extratos em suas respectivas CMNC e avaliada com base na redução do título viral e expressos em porcentagem de inibição. Os extratos aquosos de Peumus boldus e Solanum paniculatum apresentaram atividade antiviral sobre o SuHV-1 com 98% de inibição viral enquanto o de Peumus boldus inibiu apenas o BoHV-1 em 99%.

Schmallenberg Virus in Central Nervous System of Ruminants

Schmallenberg Virus in Central Nervous System of Ruminants

Immunogenicity of Lactobacillus-expressing VP2 and VP3 of the infectious pancreatic necrosis virus (IPNV) in rainbow trout

Infectious pancreatic necrosis virus (IPNV) infects salmonid fish with high mortality and causes serious economic losses to salmonid aquaculture. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, Lactobacilli/ Escherichia coli shuttle vector pPG1 (surface-displayed) or pPG2 (secretory) with the capsid VP2 gene inserted was transformed into Lactobacillus casei to yield two recombinant strains: Lc:PG1-VP2 and Lc:PG2-VP2, respectively. Rainbow trout immunized respectively with Lc:PG1-VP2, Lc:PG2-VP2, Lc:PG1-VP3 and Lc:PG2-VP3 elicited anti-IPNV immune responses (serum IgM) via oral route. Statistical results of serum IgM titer with neutralizing activity showed that immunogenicity of Lc:PG2-VP2 was more powerful than that of Lc:PG1-VP2 ( P < 0.001), Lc:PG1-VP3 ( P < 0.001) and Lc:PG2-VP3 ( P < 0.001), which was confirmed by viral loads reduction analyzed by real-time RT-PCR in orally immunized rainbow trout after virus challenge. Comparing with negative control, rainbow trout orally dosed with Lc:PG2-VP2 resulted in ∼46-fold reduction in virus load on days 10 post viral challenge as well as Lc:PG1-VP2(∼20-fold), Lc:PG2-VP3(∼6-fold) and Lc:PG1-VP3(∼3-fold). Taken together, Lc:PG2-VP2 exhibited a more appropriate candidate as live bacteria vaccine against IPNV infection in rainbow trout.

Photocatalytic inactivation of bacteriophages by TiO2-coated glass plates under low-intensity, long-wavelength UV irradiation

The International Organization for Standardization (ISO) was used to evaluate antibacterial activity by titanium dioxide (TiO(2)) photocatalysis since 2006. We evaluated photocatalytic inactivation of Qβ and T4 bacteriophages induced by low-intensity, long-wavelength ultraviolet A (UVA; 0.1 mW cm(-2) and 0.001 mW cm(-2)) irradiation on a TiO(2)-coated glass plate using the ISO methodology. The results indicated that both bacteriophages were inactivated at 0.001 mW cm(-2) UVA. The intensity of UV light, including long-wavelength light (UVA), is very low in an actual indoor environment. Thus, TiO(2) photocatalysis can be beneficial for inactivating viruses in an indoor environment. Experiments using qPCR and bovine serum albumin degradation assume that viral inactivation is caused by outer viral protein disorder and not by viral RNA reduction by reactive oxygen species produced during TiO(2) photocatalysis. Furthermore, we showed that the ISO methodology for standard testing of antibacterial activity by TiO(2) photocatalysis can be applied to assess antiviral activity.

Prediction of sustained response to low‐dose pegylated interferon alpha‐2b plus ribavirin in patients with genotype 1b and high hepatitis C virus level using viral reduction within 2 weeks after therapy initiation

Continuation of pegylated interferon (PEG-IFN) plus ribavirin at the recommended dose is difficult in elderly patients and/or patients with cytopenia or complications. Whether the therapeutic efficacy of low-dose PEG-IFN plus ribavirin therapy could be predicted based on virological response within 2 weeks of therapy initiation was evaluated. A total of 106 patients with a high viral load of genotype-1b hepatitis C virus (HCV) underwent low-dose PEG-IFN plus ribavirin therapy. PEG-IFN alpha 2b (0.75 µg/kg per week) and ribavirin (600-800 mg/day) were administered for 48 weeks. Sustained virological response (SVR) was achieved in 37%, and treatment was discontinued in 9%. On univariate analysis of SVR-contributing factors, significant differences were noted in the white blood cell count, platelet count, fibrosis markers, and viral reduction within 2 weeks from therapy initiation. On multivariate analysis, the platelet count and the reduction in the HCV core antigen level at week 2 were independent factors. The positive predictive value (PPV) and the negative predictive value (NPV) for SVR based on a 1-log or greater HCV-RNA level reduction at week 2 were 65% and 90%, respectively, and those based on HCV core antigen level at week 2 were 64% and 97%, respectively. PPV and NPV based on a 2-log or greater reduction of the RNA level were 86% and 67%, respectively, and those based on the core antigen level were 93% and 69%, respectively. Evaluation of viral reduction at week 2 after therapy initiation is useful for predicting SVR to low-dose PEG-IFN plus ribavirin therapy.

Effects of Ilex paraguariensis A. St. Hil. (Yerba Mate) on Herpes Simplex Virus Types 1 and 2 Replication

The antiherpes effects of the crude extract obtained from Ilex paraguariensis leaves (yerba mate) and their purified fractions were investigated. The most active fraction was selected and assayed to determine the viral multiplication steps upon which it acted. In order to detect the major components of this fraction, thin layer chromatography (TLC) analysis was performed. The antiviral activity was evaluated against HSV-1 and HSV-2 by a viral plaque number reduction assay (IC(50) ) and the cytotoxicity by a MTT assay (CC(50) ). According to the obtained results, all tested samples showed antiherpes activity at noncytotoxic concentrations, and the ethyl acetate fraction was the most active (SI = CC(50) /IC(50) = 188.7 and 264.7 for HSV-1 and HSV-2, respectively). The results also demonstrated that this fraction exerts antiviral activity by the reduction of viral infectivity, the inhibition of virus entry into cells and cell-to-cell virus spread, as well as by the impaired levels of ICP27, ICP4, gD and gE proteins of HSV-1. The TLC analysis showed that this fraction contains monodesmosidic triterpenoid saponins, matesaponin-1 (a bidesmosidic one), caffeic and chlorogenic acids and rutin, which suggests that they could act synergistically and be responsible for the detected antiherpes activity.

Macrophage Endocytic Trafficking of Antiretroviral Nanoparticles

Nanoformulated antiretroviral therapy can improve drug compliance for people infected with HIV. Additional benefits would include specific drug deliveries to viral reservoirs and reduction in systemic toxicities. In this article, we describe mechanisms of crystalline antiretroviral nanoparticle (NP) uptake, intracellular trafficking and release in human monocyte-derived macrophages. Following clathrin-dependent endocytosis NPs bypassed lysosomal degradation by sorting from early endosomes to recycling endosome pathways. Disruption of this pathway by siRNAs or brefeldin-A impaired particle release. Proteomic and biological analysis demonstrated that particle recycling was primarily Rab11 regulated. Particles were released intact and retained complete antiretroviral efficacy. These results suggest possible pathways of subcellular transport of antiretroviral nanoformulations that preserve both particle integrity and antiretroviral activities demonstrating the potential utility of this approach for targeted drug delivery.

Time-Dependent Effects of Pomegranate Juice and Pomegranate Polyphenols on Foodborne Viral Reduction

Pomegranate juice (PJ) and pomegranate polyphenolic extracts (PP) have antiviral effects against HIV-1, influenza, herpes, and poxviruses, and we recently demonstrated their effect against human noroviral surrogates. In the present study, the time-dependent effects of two commercial brands of PJ and PP at two concentrations (2 and 4 mg/mL) on the infectivity of foodborne viral surrogates (feline calicivirus FCV-F9, murine norovirus MNV-1, and MS2 bacteriophage) at room temperature for up to 1 h were evaluated. Each virus at ∼5 log(10) plaque-forming units (PFU)/mL was mixed with equal volumes of PJ, or PP at 4 or 8 mg/mL, and incubated for 0, 10, 20, 30, 45, and 60 min at room temperature. Viral titers after each treatment were determined by standardized plaque assays and compared with untreated controls. Virus titer reduction by PJ and PP was found to be a rather rapid process, with ≥50% of titer reduction occurring within the first 20 min of treatment for all three tested viruses. Within the first 20 min, titer reductions of 3.12, 0.79, and 0.23 log(10) PFU/mL for FCV-F9, MNV-1, and MS2, respectively, were obtained using PJ. FCV-F9, MNV-1, and MS2 titers were reduced by 4.02, 0.68, and 0.18 log(10) PFU/mL with 2 mg/mL PP and 5.09, 1.14, and 0.19 log(10) PFU/mL with 4 mg/mL PP, respectively, after 20 min. The mechanism of viral reduction by PJ and PP needs to be elucidated and clinical trials should be undertaken before recommending for therapeutic or preventive purposes.