Qualification of a novel inline spiking method for virus filter validation

Virus filters are widely used in bioprocessing to reduce the risk of virus contamination in therapeutics. The small pores required to retain viruses are sensitive to plugging by trace contaminants and frequently require inline adsorptive prefiltration. Virus spiking studies are required to demonstrate virus removal capabilities of the virus filter using scale down filters. If prefiltration removes viruses and interferes with the measurement of virus filter LRV, the standard approach is to batch prefilter the protein solution, spike with virus, and then virus filter. For a number of proteins, batch prefiltration leads to increased plugging and significantly lower throughputs than inline prefiltration. A novel inline spiking method was developed to overcome this problem. This method allows the use of inline prefiltration with direct measurement of virus filter removal capabilities. The equipment and its operation are described. The method was tested with three different protein feeds, two different parvovirus filters, two virus injection rates; a salt spike, a bacteriophage spike, and two mammalian virus spikes: MMV and xMuLV. The novel inline method can reliably measure LRV at throughputs representative of the manufacturing process. It is recommended for applications where prefiltration is needed to improve throughput, prefiltration significantly reduces virus titer, and virus filter throughput is significantly reduced using batch vs. inline prefiltration. It can even help for the case where the virus preparation causes premature plugging.     

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10¹⁰ PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4‐5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3‐101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79‐85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic‐Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V₇₅). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less‐pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log₁₀. A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V₇₅ for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:135–144, 2015     

Microscopic visualization of virus removal by dedicated filters used in biopharmaceutical processing: Impact of membrane structure and localization of captured virus particles

Virus filtration with nanometer size exclusion membranes (“nanofiltration”) is effective for removing infectious agents from biopharmaceuticals. While the virus removal capability of virus removal filters is typically evaluated based on calculation of logarithmic reduction value (LRV) of virus infectivity, knowledge of the exact mechanism(s) of virus retention remains limited. Here, human parvovirus B19 (B19V), a small virus (18–26 nm), was spiked into therapeutic plasma protein solutions and filtered through Planova™ 15N and 20N filters in scaled-down manufacturing processes. Observation of the gross structure of the Planova hollow fiber membranes by transmission electron microscopy (TEM) revealed Planova filter microporous membranes to have a rough inner, a dense middle and a rough outer layer. Of these three layers, the dense middle layer was clearly identified as the most functionally critical for effective capture of B19V. Planova filtration of protein solution containing B19V resulted in a distribution peak in the dense middle layer with an LRV >4, demonstrating effectiveness of the filtration step. This is the first report to simultaneously analyze the gross structure of a virus removal filter and visualize virus entrapment during a filtration process conducted under actual manufacturing conditions. The methodologies developed in this study demonstrate that the virus removal capability of the filtration process can be linked to the gross physical filter structure, contributing to better understanding of virus trapping mechanisms and helping the development of more reliable and robust virus filtration processes in the manufacture of biologicals.     

Performance of a novel Viresolve NFR virus filter

Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters. Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.     

Particle-based analysis elucidates the real retention capacities of virus filters and enables optimal virus clearance study design with evaluation systems of diverse virological characteristics

In virus clearance study (VCS) design, the amount of virus loaded onto the virus filters (VF) must be carefully controlled. A large amount of virus is required to demonstrate sufficient virus removal capability; however, too high a viral load causes virus breakthrough and reduces log reduction values. We have seen marked variation in the virus removal performance for VFs even with identical VCS design. Understanding how identical virus infectivity, materials and operating conditions can yield such different results is key to optimizing VCS design. The present study developed a particle number-based method for VCS and investigated the effects on VF performance of discrepancies between apparent virus amount and total particle number of minute virus of mice. Co-spiking of empty and genome-containing particles resulted in a decrease in the virus removal performance proportional to the co-spike ratio. This suggests that empty particles are captured in the same way as genome-containing particles, competing for retention capacity. In addition, between virus titration methods with about 2.0 Log₁₀ difference in particle-to-infectivity ratios, there was a 20-fold decrease in virus retention capacity limiting the throughput that maintains the required LRV (e.g., 4.0), calculated using infectivity titers. These findings suggest that ignoring virus particle number in VCS design can cause virus overloading and accelerate filter breakthrough. This article asserts the importance of focusing on virus particle number and discusses optimization of VCS design that is unaffected by virological characteristics of evaluation systems and adequately reflect the VF retention capacity.     

Development and qualification of a novel virus removal filter for cell culture applications

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Use of gas chromatography-mass spectrometry versus the limulus amebocyte lysate test for the determination of airborne endotoxin in confined swine buildings

Airborne endotoxin (lipopolysaccharide, LPS) in filter samples collected in three different swine confinement buildings was determined by using the Limulus amebocyte lysate test and by applying gas chromatography-mass spectrometry (GC-MS) to analyze 3-hydroxy fatty acids (3-OH-FAs). The amounts of LPS as shown by GC-MS were 30–50 times larger than the amounts detected by the Limulus test. GC-MS revealed that 21% of the LPS collected on cellulose acetate filters and 26% on polycarbonate filters remained on the filters after buffer extraction. Better correlation with the Limulus test and the 3-OH-FA measurements was achieved when considering the sum of 3-OH C12:0 and 3-OH C14:0 rather than the sum of all of the detected 3-OH-FAs (i.e. those with 12–18-carbon chains), indicating that the bioactivity of the LPS was dependent upon the relative distribution of the 3-OH-FAs. Linear regression analysis between air concentrations of dust and endotoxin/LPS gaveR ²values that varied from 0.407 to 0.739. The air concentrations of LPS were lower in the uninsulated swine house (a green house with an alternative housing system) than in the two insulated buildings (conventional housing systems; one designed as a climate chamber, i.e. climatic parameters could be controlled), whereas the opposite was found for the concentrations of LPS in airborne dust from the three buildings. The numbers of viable bacteria and fungi were highest in the uninsulated swine house. Moreover the relative distribution of 3-OH-FAs in that building differed from the distribution in the insulated buildings, which reflects differences in the microflora, probably mainly due to differences between the housing systems and the design of the buildings.     

Virus removal from factor IX by filtration: Validation of the integrity test and effect of manufacturing process conditions

Virus removal from a high purity factor IX, Replenine^®-VF, by filtration using a Planova 15N filter has been investigated. A wide range of relevant and model enveloped and non-enveloped viruses, of various sizes, were effectively removed by this procedure. Virus removal was confirmed to be effective when different batches of filter were challenged with poliovirus-1. It was confirmed that intentionally modified filters that failed the leakage test had completely lost the ability to remove virus, thus confirming that this test demonstrates gross filter failure. In the case of the more sensitive integrity test based on gold particle removal, it was found that a pre-wash step was not essential. Planova filters that had been modified by sodium hydroxide treatment to make them more permeable, and filters manufactured with varying pore-sizes over the range of 15–35 nm, were tested. The integrity test value that resulted in the removal of >4 log₁₀ of poliovirus-1 from the product correlated with that recommended by the filter manufacturer. Virus removal from the product was not influenced by filter load mass, flow-rate or pressure. These studies confirm the robustness of this filtration procedure and allow suitable process limits to be set for this manufacturing step.     

Testing and evaluation of off-gas filters for bioreactors by a new bacterial aerosol challenge test method (TBAC).

A TNO bacterial aerosol challenge (TBAC) filter test rig was developed for direct assessment of the effectiveness of bioreactor off-gas filters as an alternative to routinely applied indirect wet integrity testing (IT). This TBAC test rig is based on bacterial aerosol challenging with Pseudomonas diminuta and dual monitoring by laser particle counting (LPC) and Andersen microbial sampling (AMS) of viable cells. The TBAC filter test rig is able to reproduce the various conditions encountered in fermentation processes. In experiments with several filters from one class, it was demonstrated that some filters were actually penetrated by up to 3,000 viable cells per test, despite their approval by commercially available IT test equipment. Repetitive filter use, prolonged use, and autoclaving of filters resulted in an increase in pressure drop over the filter but improved the performance of leaking/deviant filters due to building up of a filter cake (this phenomenon was identified by electron microscopy). The integrity tests used were found inadequate for accurate assessment of filter quality. Certification of filter lots by random tests of commercially available off-gas filters using sensitive direct methods such as those presented here might be advisable, as not all filters purchased were of appropriate quality.     

Impact of protein fouling on nanoparticle capture within the Viresolve® Pro and Viresolve® NFP virus removal membranes

Virus filtration is a robust size-based technique that can provide the high level of viral clearance required for the production of mammalian-derived biotherapeutics such as monoclonal antibodies. Several studies have shown that the retention characteristics of some, but not all, virus filters can be significantly affected by membrane fouling, but there have been no direct measurements of how protein fouling might alter the location of virus capture within these membranes. The objective of this study was to directly examine the effect of protein fouling by human immunoglobulin G (IgG) on virus capture within the Viresolve® Pro and Viresolve® NFP membranes by scanning electron microscopy using different size gold nanoparticles. IgG fouling shifted the capture location of 20 nm gold nanoparticles further upstream within the Viresolve® Pro filter due to the constriction and/or blockage of the pores in the virus retentive region of the filter. In contrast, IgG fouling had no measurable effect on the capture of 20 nm nanoparticles in the Viresolve® NFP membrane, and IgG fouling had no effect on the capture of larger 40 and 100 nm nanoparticles in either membrane. These results provide important insights into how protein fouling alters the virus retention characteristics of different virus filters.     

Low molecular weight substitutes for beef extract as eluents for poliovirus adsorbed to membrane filters.

Basic solutions of beef extract and casein were able to elute poliovirus adsorbed to four membrane filters with different chemical compositions. Hydrolyzed protein and individual amino acids were able to elute virus adsorbed to certain filters but were unable to elute virus adsorbed to other filters efficiently. A solution of 4 M urea buffered at pH 9 with 0.05 M lysine was able to elute greater than 60% of the virus adsorbed to each of the filters tested. Certain solutions of amino acids were capable of eluting virus adsorbed to one filter but permitted adsorption of virus to another filter with a different chemical composition. Acidic amino acids could interfere with elution of virus from membrane filters. Aromatic compounds with an amino group attached to the ring were good eluents for virus adsorbed to epoxy-fiberglass membrane filters. In contrast, aromatic compounds with other substituents were generally poor eluents.     

Numerical analysis of the hemodynamics and embolus capture of a greenfield vena cava filter

BACKGROUND: Vena Cava filters are used to prevent pulmonary embolism in patients with deep vein thrombosis who are unresponsive to anticoagulation therapy. Various filter designs exist in the market with different characteristics distinguishing them. An understanding of the characteristics of these filters is desirable in order to develop better designs. METHODS: A computational fluid dynamical study of the flow over an unoccluded stainless steel Greenfield Vena Cava filter (Boston Scientific, Watertown, MA) to determine its properties has been performed. Simulation of flow over a filter placed axisymmetrically in a rounded inferior vena cava has been performed at a Reynolds numbers of 1000 and the consequences of the flow (by studying parameters like shear stress and stagnation zones) have been discussed. Furthermore, a new finite element based numerical method has been developed that allows the study of capturing properties of Inferior Vena Cava filters. The key idea is the introduction of a thin-wire-model (TWM) that enables a drastic reduction in the computational cost while still maintaining control on the physics of the problem. This numerical technique has been applied to evaluate the embolus capture characteristic of a Greenfield filter. RESULTS: The flow around the unoccluded filter is found to be steady and laminar at the conditions studied. A recirculation/stagnation zone develops immediately downstream of the filter head. This zone is significantly larger when the central hole is occluded. The shear stress and stagnation zone properties for such a flow over a Greenfield filter are compared with existing literature (in vitro studies). A graph showing the regions wherein clots escape or get captured has been determined by a means of numerical simulations. The data has further been analyzed to determine the probability of clot capture as function of the clot size. CONCLUSIONS: The stagnation zone formed behind the head of the Greenfield filter is found to be smaller in size when compared to that of the same filter with the central hole occluded. A map of the shear stress distribution shows a small region having the potential for thrombogenesis. The non-Newtonian properties of blood are not seen to cause much variation in the flow field when compared to the Newtonian model. However variation in the cava size leads to a significant change in the shear stresses. This study also establishes a novel method wherein computational means are used to determine the efficacy of clot capturing of filters. These techniques can further be used to compare the different characteristics among filters.     

Internal virus polarization model for virus retention by the Ultipor® VF Grade DV20 membrane

Several recent studies have reported a decline in virus retention during virus challenge filtration experiments, although the mechanism(s) governing this phenomenon for different filters remains uncertain. Experiments were performed to evaluate the retention of PP7 and PR772 bacteriophage through Ultipor VF Grade DV20 virus filters during constant pressure filtration. While the larger PR772 phage was fully retained under all conditions, a 2‐log decline in retention of the small PP7 phage was observed at high throughputs, even under conditions where there was no decline in filtrate flux. In addition, prefouling the membrane with an immunoglobulin G solution had no effect on phage retention. An internal polarization model was developed to describe the decline in phage retention arising from the accumulation of phage in the upper (reservoir) layer within the filter which increases the challenge to the lower (rejection) layer. Independent support for this internal polarization phenomenon was provided by confocal microscopy of fluorescently labeled phage within the membrane. The model was in good agreement with phage retention data over a wide range of phage titers, confirming that virus retention is throughput dependent and supporting current recommendations for virus retention validation studies. These results provide important insights into the factors governing virus retention by membrane filters and their dependence on the underlying structure of the virus filter membrane. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:856–863, 2014     

Achieving high mass‐throughput of therapeutic proteins through parvovirus retentive filters

Parvovirus retentive filters that assure removal of viruses and virus‐like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size‐based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass‐throughput values of 7.3, 26.4, and 76.2 kg/m² were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass‐throughput values of 73, 137, and 192 kg/m² were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large‐scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small‐scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large‐scale filter capacity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Efficacy of antimicrobial filter treatments on microbial colonization of air panel filters

AIMS: To assess the activity of biostatic agents on the microbial colonization of panel filters. METHODS AND RESULTS: Microfibre glass acrylic filters, both used and unused, were examined for the presence of microorganisms. Test strains were used to verify microbial colonization of filter media. Antimicrobial agents were applied to the filter media and tested for their ability to reduce microbial colonization. The integrity of the panel filters and the antimicrobial activity trends of the filter media treated with antimicrobials were verified. A filtration efficiency test was carried out on the treated filters to evaluate filtration performance. Filters treated with antimicrobials demonstrated markedly less microbial colonization (density and varieties of species), higher filtration efficiency and delayed deterioration of the filter. CONCLUSIONS: The most important results of this study are the demonstration of preservation of the integrity of the filters and the lower release of microorganisms from treated filters. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the resolution of problems concerning the microbial contamination of panel filters in the heating, ventilating and air-conditioning systems commonly used in the electronic industry, pharmaceutical industry, hospitals and other environments where the absence of contaminating particles and microorganisms is required.     

Flow‐dependent entrapment of large bioparticles in porous process media

The need for purification of biomolecules extends to larger bioparticles as well. For example, virus purification is required for production of many vaccines and gene delivery vectors, and understanding virus removal in porous media is also important in downstream processing of therapeutic proteins and in purification of water in soils. A convective entrapment mechanism for retention of large bioparticles is discussed here based on retention of such bioparticles in pore constrictions at high enough flow rates, even under non‐binding conditions. A simple equation to predict whether such entrapment is expected to occur in a given system is derived based on a Péclet number that is proportional to the flow rate and to the cube of the bioparticle diameter. To test the theory, adenovirus was spiked onto chromatographic beds. As expected from the theory, under non‐interacting conditions a progressively larger amount of virus becomes trapped with increasing flow rate. The entrapment is reversible upon flow rate reduction, which, within the proposed model, is based on the possibility of diffusive escape from pore constrictions. This mechanism can be exploited for virus purification or removal, and the theory is also consistent with the anecdotal evidence that monoliths and membranes are more difficult to clean than conventional chromatographic beds, especially at high flow rates. Biotechnol. Bioeng. 2009; 104: 127–133 © 2009 Wiley Periodicals, Inc.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.     

Coat Protein-derived Small Particles in a Tombusvirus from River Lato

A new tombusvirus cleariy distinguishable from other members of the group was isolated in Apulia from river Lato and therefore referred to as Lato River Tombusvirus (LRV). Purified virus preparations after storage for 5 months at 4°C in 20 mM phosphate buffer, pH 7.2, contained variable amounts of small particles (c. 17 nm in diameter) in addition to typical virus particles (c. 30 nm in diameter). LRV small particles were constituted by a single protein species with a tnol. wt of 29,000, did not contain nucleic acid and were not infective but proved to be serologically undistinguishable from ordinary virus particles. Attempts to artificially produce small particles by chemical orenzymatic treatments of full size LRV virions failed but their spontaneous production nder particularstorage conditions seems to be a remarkable feature of this virus as it does not occur with other definitive tombusviruses.     

Concentration of enteroviruses from estuarine water.

Pleated cartridge filters readily adsorb viruses in estuarine water at low pH containing aluminum chloride. Adsorbed viruses are efficiently recovered by treating filters with glycine buffer at high pH. By using these procedures, it was possible to recover approximately 70% of the poliovirus added to 400 liters of estuarine water in 3 liters of filter eluate. Reconcentration of virus in the filter eluate in small volumes that are convenient for viral assays was more difficult. Reconcentration methods described previously for eluates from filters that process tap water or treated wastewater were inadequate when applied to eluates from filters used to process estuarine water containing large amounts of organic compounds. Two methods were found to permit efficient concentration of virus in filter eluates in small volumes. In both methods, virus in 3 liters of filter eluate was adsorbed to aluminum hydroxide flocs and then recovered in approximately 150 ml of buffered fetal calf serum. Additional reductions in volume were achieved by ultrafiltration or hydroextraction. By using these procedures 60 to 80% of the virus in 3 liters of filter eluate could be recovered in a final volume of 10 to 40 ml.