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The growing interest in process intensification and continuous processing has led to concerns regarding the performance of virus removal filters at low permeate fluxes. This paper presents a quantitative analysis of virus retention by the Viresolve® Pro membrane during constant permeate flux filtration and in response to process disruptions using ΦX-174, a bacteriophage, as a model for a mammalian parvovirus. Virus retention by a single layer of Viresolve® Pro membrane was reduced at lower permeate fluxes due to the greater diffusive mobility of phage, although the expected retention for a commercial device employing two layers of membrane remained above 4-logs. The retention data were well correlated with the ΦX-174 Péclet number for filtrate flux from 5 to 100 L/m2/h. Process disruptions caused an immediate transient increase in phage transmission, although this effect was much less pronounced at low permeate flux. The experimental data for phage retention, both with and without process disruptions, were well-described using an internal polarization model with the fraction of virus remaining mobile within the filter scaling as 1/ Pe 2. In contrast, the fraction of previously captured virus that were re-mobilized after a process interruption was independent of the flow rate. Model equations were developed for rapid estimation of virus performance behavior with the Viresolve® Pro membrane, facilitating the design and implementation of virus filtration processes operating at constant filtrate flux. [Display omitted] • Virus retention by the Viresolve® Pro filter reduced at low filtrate flux due to virus diffusion. • Log reduction value (LRV) well-correlated with the virus Péclet number. • Virus transmission increases immediately after process disruption. • LRV data well described using internal polarization model. • Fraction of previously captured virus re-mobilized after disruption independent of filtrate flux. [ABSTRACT FROM AUTHOR]

Abstract: Since mid-December 2011, samples from malformed lambs and calves are sent to CODA-CERVA in Belgium for diagnosis of Schmallenberg virus (SBV), a novel Orthobunyavirus that was first detected by researchers of the Friedrich-Loeffler-Institut (FLI, Germany) in German cattle in autumn 2011 and was later shown to be involved in congenital malformations in lambs, goat kids and calves. Surprisingly, by making use of real time RT-PCR (rRT-PCR) assays developed by the FLI, presence of SBV RNA could only be confirmed in part of the SBV suspected newborns examined. To investigate possible causes for non-confirmation by rRT-PCR, a comparative analysis between different organs and tissues (cerebrum, cerebellum, brain stem, spinal cord, thymus, spleen, lymph nodes, meconium) originating from respectively 90 and 81 malformed lambs and calves was undertaken. Furthermore, thoracic fluids of respectively 55 malformed lambs and calves were examined by a virus neutralization test (VNT) to evaluate the presence of neutralizing anti-SBV antibodies in these animals. Our results show that among the different organs tested by rRT-PCR, brain stem material is the most appropriate tissue for SBV detection while it could also be detected in all other tissues but to a more variable degree. The VNT test showed that 95% of the malformed lambs were positive for anti-SBV neutralizing antibodies while this was only the case for 44% of malformed calves. These immunological data suggest that a humoral immune response could assist in the clearance of SBV from the fetus during gestation and that SBV specific antibody testing should be considered together with rRT-PCR analysis for confirmation of SBV infection. [Copyright &y& Elsevier]

Abstract: Infectious bursal disease (IBD) is a highly contagious disease of chickens which leads to immunosuppression. In our previous study it was demonstrated that, possibly, CD4+ and CD8+ T cells may employ perforin and granzyme-A pathway for the clearance of IBDV-infected bursal cells. In this study, we evaluated the cytotoxic T cell responses involving two independently functioning but complementary mechanisms: Fas–Fas ligand and perforin–granzyme pathways in IBDV-infected chickens. As demonstrated previously, infection of chickens with IBDV was accompanied by influx of CD8+ T cells in the bursa and spleen. There was an upregulation in the gene expression of cytolytic molecules: Fas and Fas ligand (FasL), perforin (PFN) and granzyme-A (Gzm-A) in bursal and in the splenic tissues of IBDV inoculated chickens. Additionally, for the first time, we detected Fas, Fas ligand, Caspase-3 and PFN producing CD8+ T cells in the bursa and spleen of IBDV-infected chickens. The infiltration and activation of CD8+ T cells was substantiated by the detection of Th1 cytokine, IFN-γ. These data suggest that T cells may be involved in the clearance of virus from the target organ bursa and peripheral tissues such as spleen. The findings of these studies provide new insights into the pathogenesis of IBD and provide mechanistic evidence that the cytotoxic T cells may act through both Fas–FasL and perforin–granzyme pathways in mediating the clearance of virus-infected cells. [Copyright &y& Elsevier]

Abstract: Infectious bursal disease (IBD) is an economically important immunosuppressive disease of chickens. The IBD virus (IBDV) actively replicates in B cells and causes severe bursal damage. Generally, T cells are refractory to infection with IBDV but are known to promote virus clearance. However, the mechanisms of T cell mediated viral clearance are not well understood. In this study, we evaluated the molecular mechanisms of cytotoxic T cell responses in the pathogenesis of IBD in chickens. Infection of chickens with IBDV was accompanied by the infiltration of CD4+ and CD8+ T cells in the bursa. There was an upregulation in the gene expression of important cytolytic molecules; perforin (PFN), granzyme-A (Gzm-A), DNA repair and apoptotic proteins; high mobility proteins group (HMG) and poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF) whereas expression of NK (natural killer) lysin was downregulated. Importantly, PFN producing CD4+ and CD8+ T cells were also detected in the bursa of IBDV-infected chickens by immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was also strongly upregulated, suggesting the activation of T cells. The findings of this study highlight the mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the clearance of virus-infected cells. [Copyright &y& Elsevier]

Abstract: Virus filtration membranes are used to obtain virus clearance during the purification of biopharmaceutical products. These direct flow (also referred to as dead end or normal flow) filtration membranes are designed to reject virus particles and yield >98% product recovery for proteins less than 170kDa. Virus filtration feed streams generally have high purity and high product concentrations. Decrease in permeate flux during virus filtration, which reduces filter capacity, is most likely due to fouling by small aggregates of the product species present in the feed stream. Retrovirus and parvovirus filtration membranes, as well as two ultrafiltration membranes with NMWCOs of 300 and 10kDa (kgmol−1), were tested using feed streams consisting of minute virus of mice (MVM) in the presence and absence of 1% (w/v) BSA. A novelty of our work comes from the fact that we directly compare the performance of these membranes using realistic model feed streams consisting of MVM (an FDA recommended model virus) and BSA. This approach provides an industrially relevant benchmark of the engineering performance of virus filters with respect to a limited set of structural and operational variants. All asymmetric membranes were operated in direct flow, constant pressure mode with the filtration surface (i.e. skin surface) facing away from the feed stream as is industrial practice for virus filtration. In addition, the 10kDa ultrafiltration membrane was also operated with the skin surface facing the feed stream as is industrial practice for ultrafiltration. While little rejection of virus particles was observed for the 10kDa membrane when the skin surface faced away from the feed stream, significant (in excess of 3 log) virus rejection was observed when the skin surface faced the feed stream. Changes in permeate flux with filtrate volume were determined along with parvovirus rejection. Decreases in permeate flux were shown to result from fouling in the presence of BSA. No decrease in permeate flux was observed in the absence of BSA. The decrease in permeate flux has been analyzed in terms of classical pore blockage models. The results of this work, which compare the performance of virus filtration and similar ultrafiltration membranes, provide insights into designing virus filtration membranes. [Copyright &y& Elsevier]

Abstract: In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50mM NaOH/0.5M Na2SO4 with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance. [Copyright &y& Elsevier]

Abstract: Background: Measles virus (MV) RNA was detected 1 month after hospitalization with measles in more than half of Zambian children but the duration of detectable RNA was not determined. Objectives: To characterize the time course of MV clearance and identify factors associated with presence of viral RNA at late times after clinical recovery from infection. Study design: Blood, urine and nasopharyngeal specimens from 49 Zambian children with laboratory-confirmed measles were collected a median of 100 days (range 65–118) after rash onset. Samples were assayed for MV nucleocapsid and hemagglutinin RNA by reverse transcriptase–polymerase chain reaction. Amplified products were sequenced. Selected immunologic studies were performed. Results: MV RNA was detected in at least one specimen from 18 children (37%). Eighteen percent of 44 blood mononuclear cell, 23% of 30 nasopharyngeal and 50% of 6 urine specimens were positive. Detection was not associated with HIV-1 infection, % CD4+ T lymphocytes, plasma interleukin-10 levels or persistent MV-specific IgM. The MV genotype was D2 and sequences of late specimens were the same as specimens collected during acute illness. Conclusions: Presence of viral RNA at multiple sites more than 3 months after acute disease suggests that clearance of MV-infected cells occurs over many months. [Copyright &y& Elsevier]

Abstract: Objective: Proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumour viruses. The E7 oncoprotein of high-risk human papillomavirus (HPV) is known to activate PCNA, shown to be up-regulated in CIN and cervical cancer (CC), but still incompletely studied as an intermediate endpoint marker in this disease. Material and methods: As part of our HPV-PathogenISS study, a series of 150 CCs and 152 CIN lesions were examined using immunohistochemical (IHC) staining for PCNA, and tested for HPV using PCR with three primer sets (MY09/11, GP5+/GP6+, SPF). Follow-up data were available from all SCC patients, and 67 of the CIN lesions had been monitored with serial PCR for HPV after cone treatment. Results: Expression of PCNA increased in parallel with the grade of CIN, with major up-regulation upon transition to CIN3 (OR 21.77; 95%CI 6.59–71.94) (p =0.0001). Intense PCNA expression was 100% specific indicator of CIN, with 100% PPV, but suffers from low sensitivity (34.8%) and NPV (10.8%). PCNA expression was also significantly associated to HR-HPV with OR 3.02 (95%CI 1.71–5.34) (p =0.0001), and this association was not confounded by the histological grade (Mantel–Haenszel common OR=2.03; 95%CI 1.06–3.89) (p =0.033). Expression of PCNA did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic predictor in CC in univariate or in multivariate analysis. Conclusions: Up-regulation of PCNA was closely associated with HR-HPV and progressive CIN, most feasibly explained by the abrogation of normal cell cycle control by the E7 ongogene, reverting the p21Cip1-mediated inhibition of PCNA. However, the fact that PCNA is also expressed in normal squamous epithelium precludes the use of this marker as a potential screening tool for CC. [Copyright &y& Elsevier]

The impact of corticosteroid treatment on virological course of coronavirus disease 2019 (COVID-19) patients remains unclear. This study aimed to explore the association between corticosteroid and viral clearance in COVID-19. The clinical data of COVID-19 patients from 10 hospitals of Jiangsu, China, were retrospectively collected. Cox regression and Kaplan–Meier analysis were used to analyze the adverse factors of virus clearance. Of the 309 COVID-19 patients, eighty-nine (28.8%) patients received corticosteroid treatment during hospitalization. Corticosteroid group showed higher C-reactive protein (median 11.1 vs. 7.0 mg/l, P = 0.018) and lower lymphocytes (median 0.9 vs. 1.4 × 109/l, P < 0.001) on admission. Fever (93.3% vs. 65.0%, P < 0.001) and cough (69.7% vs. 57.3%, P = 0.043) were more common in corticosteroid group. The proportions of patients with severe illness (34.8% vs. 1.8%, P < 0.001), respiratory failure (25.8% vs. 1.4%, P < 0.001), acute respiratory distress syndrome (4.5% vs. 0%, P = 0.002), and admission to ICU (20.2% vs. 0.9%, P < 0.001) were significantly higher in corticosteroid group than non-corticosteroid group. The duration of virus clearance (median 18.0 vs. 16.0 days, P < 0.001) and hospitalization (median 17.0 vs. 15.0 days, P < 0.001) were also significantly longer in corticosteroid group than non-corticosteroid group. Treated with corticosteroid (Hazard ratio [HR], 0.698; 95% confidence interval [CI], 0.512 to 0.951; P = 0.023) was an adverse factor of the clearance of SARS-CoV-2, especially for male patients (HR, 0.620; 95% CI, 0.408 to 0.942; P = 0.025). The cumulative probability of SARS-CoV-2 clearance was lower in corticosteroid group (P < 0.001). Corticosteroid treatment may delay the SARS-CoV-2 clearance of COVID-19 patients and should be used with cautions. [ABSTRACT FROM AUTHOR]