Concentrated Virus Preparations Research Articles

A simple and efficient method for purification of intact white spot syndrome virus (WSSV) viral particles

A new simple and efficient method for isolation of intact WSSV viral particles from infected crayfish tissues with high yield was developed. Abundant viral particles could be obtained with only a few steps of conventional differential centrifugations, while no density gradient centrifugation or ultracentrifugation was required. The concentrated virus preparations were further studied by transmission electron microscopy and polyacrylamide gel electrophoresis. Using negative-staining TEM, we found that purified viral particles were coated with integral envelope. At least 23 major structural proteins from purified WSSV virions could be observed by SDS-PAGE. By this method, about 10 12 viral particles could be recovered from 10 g of infected crayfish tissues. Moreover, purified virus does not lose its biological activity. Using purified virus, the minimal amount of WSSV that could initiate a successful virus proliferation in crayfish was determined.

Concentration of plant viruses using monolithic chromatographic supports

A new chromatographic medium, Convective Interaction Media ® (CIM) disk monolithic columns, was applied to plant virus concentration. The ability of the columns to concentrate highly diluted plant viruses was tested on a model plant virus, rod-shaped tomato mosaic virus (ToMV). Enzyme-linked immunosorbent assay (ELISA) was used for the quantitative analysis. The virus was concentrated using a strong anion exchanger, CIM quaternary amine (QA) disk monolithic column. A high salt concentration was used to elute the concentrated virus from the columns. It has been demonstrated that ToMV, which had been diluted considerably below the sensitivity of ELISA, was concentrated by several orders of magnitude in the one-step procedure. Concentrated virus preparations could be used directly for ELISA testing. In comparison with methods described for concentrating plant viruses from irrigation water, the above procedure may provide a much faster and more efficient way to concentrate highly diluted plant viruses. The procedure could be applied to the testing of other highly diluted plant viruses, and to concentrating viruses for antiserum production.

Tomato yellow leaf curl geminivirus: serological evaluation of an improved purification method 1

Tomato yellow leaf curl geminivirus is an important limiting factor for tomato production and is now expanding into new areas. Difficulties in virus purification and low antigenicity of virus particles have so far allowed the production only of low‐titre antisera. To obtain better antisera, the purification method has to be improved, and this in turn requires better methods for measuring the yields of virus obtained using alternative purification procedures. For this purpose, we have investigated four types of ELISA test. Among these, biotin‐avidin ELISA gave good results under all conditions; indirect ELISA gave better results with concentrated virus preparations but did not work with crude extracts. Two DAS‐ELISA types, differing in conjugation procedures, gave less reliable results. On the bases of these tests, the best purification method proposed included a 24‐h incubation of the homogenate at 37°C with Driselase (macerating enzymes), and gave a 4–8 fold increase in yield, compared with the best method previously used. Nicotiana benthamiana was confirmed to be the best source plant.

Purification of Barley Yellow Streak Mosaic Virus and Detection by DAS-ELISA and ISEM Using Polyclonal Antibodies

Skaf, J. S., and Carroll, T. W. 1995. Purification of barley yellow streak mosaic virus and detection by DAS-ELISA and ISEM using polyclonal antibodies. Plant Dis. 79:1003-1007. A purification procedure was developed for barley yellow streak mosaic virus (BaYSMV), the causal agent of a disease in barley and wheat. The procedure utilizes differential and density gradient centrifugation in Percoll gradients and yields highly concentrated virus preparations. These preparations were infectious in Nicotiana benthamiana. Electron microscopy of non-glutaraldehyde-fixed virus particles negatively stained or detergent-treated revealed the presence of a finely granular viral nucleocapsid and an envelope presumed to be lipoidal in nature. Internal cross striations characteristic of rhabdovirus particles were not observed. Concentrated virus preparations were used to produce rabbit polyclonal antibodies that were useful in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) for the detection of the virus in barley, wheat, N. benthamiana, as well as in its vector, the brown wheat mite Petrobia latens. The virus was detected by ELISA and ISEM in the Pocatello Valley, Idaho, in 1992 and 1993, verifying our 1991 detection of the virus in the same valley. Leaf extracts from BaYSMV-infected barley or N. benthamiana plants injected via hypodermic needle and syringe were infectious, causing characteristic disease symptoms in barley. By contrast, viral nucleic acid preparations used to rub-inoculate N. benthamiana plants were non-infectious.

International bank for foot-and-mouth disease vaccine: stability studies with virus concentrates and vaccines prepared from them

An international bank for foot-and-mouth disease (FMD) vaccine has been established at the Pirbright Laboratory of the AFRC Institute for Animal Health. The bank is based on concentrated virus preparations stored in the gaseous phase of liquid nitrogen and is capable of producing up to 0.5 million cattle doses of each of five common strains of the virus (FMDV) for the member nations of the bank. This paper describes the initial and subsequent testing of the virus concentrates and vaccines prepared from them. There was no evidence of deterioration of the virus concentrates during liquid nitrogen storage. High levels of protection of cattle and guinea-pigs were achieved when vaccines were used immediately following preparation from the recently thawed virus concentrates. In contrast, storage of vaccines of 4°C for one or more months resulted in loss of potency in guinea-pigs and was attributed, in part to proteolytic enzymes in the virus concentrates.

The detection and inhibition of proteolytic enzyme activity in concentrated preparations of inactivated foot-and-mouth disease virus

Proteolytic enzyme activity was detected in a large number of concentrated preparations of inactivated foot-and-mouth disease virus. Several lines of evidence indicated that at least some of this activity could be attributed to BHK cells, although low levels of microbial contamination in many of our preparations could not be discounted and would certainly enhance the cellular proteolytic activity. From an experiment with different concentrations of trypsin, it was concluded that the proteolytic activities of virus concentrates were sufficient to cause significant degradation of VP1. It was also shown that Trasylol and ox serum were effective inhibitors of proteolytic enzymes in concentrated virus preparations stored at 4 degrees C for 8 months.

Attenuation of virulence of a bunyavirus involving an L RNA defect and isolation of LAC/SSH/LAC and LAC/SSH/SSH reassortants

Unlike wild-type La Crosse (LAC), or snowshoe hare (SSH) viruses, or the reassortants SSH/SSH/LAC, LAC/SSH/SSH, LAC/SSH/LAC, SSH/LAC/LAC, and SSH/LAC/SSH (large/medium/small, L/M/S, RNA genotypes), LAC/LAC/LAC and LAC/LAC/SSH reassortant bunyaviruses recovered from mixed virus infections involving Group II temperature-sensitive ( ts) mutants of SSH/LAC/SSH and a LAC virus Group I ts 16 mutant were found to be significantly less virulent after intracerebral (ic) inoculation into 4-week-old mice. Mixed virus infections involving the same SSH/LAC/SSH ts mutants and an alternate LAC Group I ts mutant (LAC I-20) gave reassortants that were almost as virulent as wild-type LAC virus. It has previously been shown that bunyavirus Group II ts mutants have defects in their L RNA gene products, while Group I ts mutants have defects in their M RNA gene products (Gentsch et al., 1977b, J. Virol. 24, 893–902). In the analyses reported in this paper it has been determined that, in addition to a ts lesion in its M RNA species, the L RNA segment of LAC I ts 16 has a defect that is responsible for attenuation of the derived LAC/LAC/LAC and LAC/LAC/SSH reassortant progeny. The attenuated viruses were found to have lower ic LD 50 to PFU ratios than nonattenuated viruses. Two new SSH-LAC reassortant viruses used in this study (namely LAC/SSH/LAC and LAC/SSH/SSH), which had previously eluded detection by standard recombination analyses with unconcentrated viruses, have been obtained from SSH II-22 × LAC I-20 ts mutant virus crosses using polyethylene glycol-6000 (PEG) concentrated virus preparations. The difficulties encountered in obtaining these reassortants using unconcentrated viruses is in contrast to the ease of recovery of other SSH-LAC reassortants from their parental ts mutant virus crosses in standard recombination assays.

Purification and biophysical properties of human coronavirus 229E

Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 10 9.0–10 9.5 TCID 50/ml and plaque titers from 10 10.2–10 10.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO 4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E 1cm 1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S 20,v 0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.

Detection of simian type-C particles in Mason-Pfizer virus infected cultures.

A simian type-C virus has been detected in cultures chronically infected with Mason-Pfizer monkey virus (M-PMV). Simultaneous budding of M-PMV and type-C virus particles from the same cells was observed in cultures incubated at 37 or 40 degrees C. However, the frequency of such cells was greater in cultures grown at 40 degrees C. Although clusters of type-C viral buds were seen at the surface of the cells, extracellular mature type-C particles in cell pellets or concentrated virus preparations were very rarely found. The increase in frequency of type-C buds was found to be transitory since cultures adapted to growing at the high temperature demonstrated budding type-C particles only occasionally. Cultures producing type-C buds were found to contain, in addition to M-PMV antigens, serological activity with polyvalent antisera produced against multiple structural components of endogenous baboon virus (BV) or simian sarcoma virus (SiSV). The reactivity, however, was found not to be serologically related to the major SiSV P28 core protein.

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol.

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.

Concentration of Rous Sarcoma Virus from Tissue Culture Fluids with Polyethylene Glycol

Concentration of Rous sarcoma virus from tissue culture fluids with polyethylene glycol, with and without NaCl or dextran sulfate, resulted in significant and highly variable losses caused by entrapment of virus particles in proteinaceous debris. Treatment of concentrated preparations with Pronase greatly enhanced the recovery of virions. Maximum recovery of virus particles was obtained by the addition of 8% polyethylene glycol and 0.4 M NaCl to tissue culture fluids, followed by Pronase treatment of the concentrated virus preparations.

Cell fusion by canine distemper virus-infected cells.

AV3 cells (continuous human amnion) infected with the Onderstepoort strain of canine distemper virus produced cell fusion within 2 to 5 hr when added to AV3 cell monolayers. An apparent requirement for intact, infected cells was demonstrated by showing that (i) frozen-and-thawed infected cells failed to induce fusion, (ii) infected cells frozen in the presence of glycerol retained their ability to induce fusion, (iii) infected cells subjected to swelling in hypotonic buffer and homogenization lost their ability to fuse cells, and (iv) semipurified and concentrated virus preparations with infectivity titers as high as 10(7.5) mean tissue culture doses per ml failed to induce fusion within 5 hr. Preparations of intact, infected cells had a mean log(10) ratio of infectivity to fusion activity of 3.6. Treatment with beta-propiolactone rendered the active preparations free from detectable infectivity while they retained their ability to cause cell fusion. Cycloheximide did not block the formation of syncytia in assay cells. This type of cell fusion was neutralized by canine distemper virus immune antisera, and measles virus immune sera showed a slight degree of cross-neutralization. Other cell lines, HEp-2, MA 139 (embryonic ferret lung), MA 104 (embryonic rhesus monkey kidney), and Vero (African green monkey kidney) were also susceptible.

Potency of Adenovirus Vaccines: Comparison of Unconcentrated, Concentrated, and Soluble Antigen Preparations

Five adenoviruses were effectively concentrated and partially purified by methanol precipitation. The soluble antigens from one adenovirus concentrate were obtained by adsorption to and elution from calcium phosphate. Formaldehyde-inactivated vaccines prepared from the virus concentrates were more antigenic in guinea pigs than a National Institutes of Health reference vaccine or one prepared from unconcentrated adenovirus. The soluble antigens also proved to be antigenic. The data indicate that adenovirus vaccines of superior potency can be prepared from concentrated virus preparations and that the extracted soluble antigens are immunogenic.

Potency of Adenovirus Vaccines: Comparison of Unconcentrated, Concentrated, and Soluble Antigen Preparations

Five adenoviruses were effectively concentrated and partially purified by methanol precipitation. The soluble antigens from one adenovirus concentrate were obtained by adsorption to and elution from calcium phosphate. Formaldehyde-inactivated vaccines prepared from the virus concentrates were more antigenic in guinea pigs than a National Institutes of Health reference vaccine or one prepared from unconcentrated adenovirus. The soluble antigens also proved to be antigenic. The data indicate that adenovirus vaccines of superior potency can be prepared from concentrated virus preparations and that the extracted soluble antigens are immunogenic.

Enterovirus Immunizing Antigens in the from of Cation-Stabilized and Concentrated Virus Preparations

Summary A method for preparation of concentrated enteroviral antigens is described. Heating virus at 50°C in M MgCl2 inactivates adventitious simian agents. Addition of 25 mM AlCl3 and 50 mM Na2CO3 to the heated, stabilized virus produces a precipitate which selectively adsorbs enteroviruses. The sediment is reconstituted in one tenth the original volume of virus. Guinea pigs injected with cation-precipitated antigens produced a higher antibody response than animals receiving untreated antigens.

Purification of barley yellow dwarf virus

A vector-specific strain of barley yellow dwarf virus (transmitted selectively by Macrosiphum avenae) was partially purified from frozen oats by differential centrifugation following clarification by chloroform. Since the virus is not mechanically transmissible to plants, infectivity assays were based on feeding aphid vectors through membranes on test inocula. Concentrated virus preparations stored at 3°C remained infectious for up to 16 weeks; similar preparations were infectious following treatment (18 hours at 15°C) with any of several proteases and nucleases. A major limiting factor in purification appeared to be the low concentration of virus in source plants; usually the yield of purified virus from 1 liter of plant juice was only about 25–50 μg. Analytical sucrose density-gradient centrifugation of preparations made from infected oats revealed a component (sedimentation coefficient 115–118 S) that was not present in comparable preparations made from healthy oats in each of three experiments. A dense polyhedral particle about 30 mμ in diameter was the major constituent of the extra component from infected plants. This polyhedral particle is believed to be the virus because it sedimented to the same depth as did the infectious entity in rate- and equilibrium-zonal centrifugations, and it migrated with the infectious entity in Sephadex 4 gel filtration. The particle also was obtained from viruliferous aphids.

Application of I131 to determination of viral antibodies.

SummaryExperiments are described in which preparations of yellow fever virus, Bwamba fever virus and influenza virus were allowed to react with corresponding antisera labeled with radioactive iodine (I131). No significant differences were observed in the activity of the sediments obtained with the first 2 virus systems from those of appropriate controls. The influenza virus-immune serum sediment showed significantly more activity than any of its control preparations. It is suggested that the technic may be of value in virus studies where relatively pure and concentrated virus preparations are available.

Failure of Aëdes Aegypti to Transmit Yellow Fever Cultured Virus (17D) 1

Summary and Conclusions 1. The quantity of virus in the circulation of a vaccinated human being is too small under most circumstances to permit the infection of Aedes aegypti. 2. Of eight human volunteers fed on by Aedes aegypti on the 6th and 7th days after vaccination, two had minimal amounts of virus on the 7th day. The mosquitoes feeding at that time were not infected. 3. Of twelve lots of mosquitoes fed on rhesus monkeys after vaccination, five lots engorged at a time when the concentration of virus in the blood stream was as great as or greater than that observed in a carefully studied group of twenty-nine human beings after vaccination. None of the five lots was infected. 4. Four attempts were made to infect Aedes aegypti in the larval stage by immersing them in large quantities of vaccine virus. The adults emerging from larvae infected in the two more concentrated virus preparations were shown, by injection into mice and monkeys, to be infected. However, neither lot was capable of transmitting the virus by bite. In one case thirty-five insects failed to transmit by bite, though the identical mosquitoes were shown to contain the virus in their bodies. In the other case twenty-nine insects gave a similar result. None of the three monkeys infected by the injection of the mosquitoes had fever, nor was there any increase in circulating virus as a result of the single passage through the bodies of the mosquitoes. 5. It is concluded that the danger of Aedes aegypti transmitting the 17D vaccine virus is at best slight, and, in all probability, nonexistent.