Purification and characterization of bovine placental retinol-binding protein

A major low mol wt acidic protein, 3B3, produced from cultures of day 29-90 bovine allantoic membranes (in the presence of [3H]leucine or [35S]methionine) and from day 29-60 allantoic fluid, has been purified. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical mol wt (23,200 +/- 900) when analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence analysis of 3B3 isolated from allantoic fluid on the first 43 amino acids showed that 3B3 had 93% and 91% homology with rabbit and human plasma retinol-binding protein (RBP), respectively. The UV absorption spectrum and the fluorescence excitation and emission spectra of purified 3B3 from both sources indicated the presence of bound retinol. Rabbit antiserum was raised against placental RBP (3B3) isolated from allantoic membrane culture medium. Placental RBP was immunoprecipitated from radiolabeled allantois and chorion culture medium and was detected in allantoic membrane culture medium and allantoic fluid by Western blotting. These results suggested that bovine placental membranes secrete RBP into allantoic fluid and that placental RBP may play important roles in vitamin A metabolism in the developing embryo.     

Virucidal activity of disinfectants. Influence of the serum protein upon the virucidal activity of disinfectants

Five disinfectants were tested for virucidal activity on three DNA viruses and three RNA viruses in the presence or absence of serum protein. Disinfectants of the aldehyde and halogen groups had a virucidal activity on human herpes virus, bovine rhabdo virus, human immunodeficiency virus, human adeno virus, porcine parvo virus, and polio virus. Disinfectants of the invert and amphoteric soap groups, and biganide group had a destructive effect on RNA and DNA viruses possessing an envelope. The presence of serum protein exerted great influence upon the virucidal activity of disinfectants of the invert and amphoteric soap groups.     

Virucidal activity of disinfectants: virucidal activities of disinfectants on some viruses coated on several materials and its durability

First, four disinfectants were tested for virucidal activity on viruses coated upon materials. Disinfectants of the aldehyde and halogen groups had a destructive effect on both enveloped and non-enveloped DNA and RNA viruses coated on a cotton gauze, stainless chips, wood shavings, polyplopylene resin chips and latex resin chips, respectively. Disinfectant of the biguanide group had a virucidal activity on both enveloped DNA and RNA viruses. Secondary, five disinfectants were tested for the durability of their virucidal activity on two DNA and RNA viruses after preparing working solutions. The disinfectants of the aldehyde group maintained their virucidal efficiency for 2 to 3 days. Disinfectants of the invert and amphoteric soap groups, the biguanide group and the halogen group maintained their virucidal efficiency for 1 to 2 days. In the presence of bovine serum, the virucidal activity of the aldehyde group and the halogen group were not influenced but that of the invert and amphoteric soap groups were strongly influenced and lost their effect.     

Inactivation of human enterovirus 71 and coxsackie virus A16 and hand, foot, and mouth disease

Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major etiological agents in major outbreaks of hand, foot, and mouth disease. Transmission of these viruses is facilitated by prolonged environmental survival and their resistance to biocides, and effective disinfection is crucial to interrupt the cycle of environmental spread. We tested the virucidal efficacy of sodium hypochlorite against both EV71 and CVA16, performed according to the Association of Official Analytical Chemists (AOAC) test criteria and methods approved by the US Environmental Protection Agency. Our results indicated the complete inactivation of infectivity of EV71 and CVA16 after a 5-minute exposure to 3120 ppm sodium hypochlorite.     

'In vitro' antiviral activity of neem (Azadirachta indica. A. Juss) leaf extract against group B coxsackieviruses

The antiviral and virucidal effect of methanolic extract fraction of leaves of neem (Azadirachta indica A. Juss) (NCL-11) was studied regarding its activity and possible mechanism of action against Coxsackie B group of viruses. NCL-11 inhibited plaque formation in 6 antigenic types of Coxsackie virus B at a concentration of 1000 micrograms/ml at 96 hrs. 'in vitro'. Additionally virus inactivation, yield reduction and effect of time of addition assays suggested that NCL-11 was most effective against coxsackie virus B-4 as a virucidal agent besides interfering at an early event of its replicative cycle. The evidence suggested that presence of a battery of compounds besides flavonoids, triterpenoids and their glycosides in NCL-11 have antiviral action for coxsackie B group of viruses 'in vitro.' The minimal inhibitory concentrations were not toxic to Vero (African green monkey kidney), cells; subtoxic concentration was 8,000 micrograms/ml and cytotoxic concentration 10,000 micrograms/ml, which was confirmed by trypan blue dye exclusion test.     

Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid.

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.     

Structure-activity relationships of dengue antiviral polycyclic quinones

The virucidal and antiviral photoactivities of three compounds, hypericin, tetrabromohypericin and gymnochrome B, were evaluated against dengue viruses. All the three products were active, and both the virucidal and antiviral activities were enhanced by light. Gymnochrome B was more potent than hypericin and tetrabromohypericin. The presence of the side chains on the hypericin core of gymnochromes appears to be beneficial for both virucidal and antiviral activities. This enhanced activity is likely to be linked to a complementary mechanism independent of photoactivation.     

Phi 6 Bacteriophage Inactivation by Metal Salts,Metal Powders,and Metal Surfaces

The interaction of phages with abiotic environmental surfaces is usually an understudied field of phage ecology. In this study, we investigated the virucidal potential of different metal salts, metal and ceramic powders doped with Ag and Cu ions, and newly fabricated ceramic and metal surfaces against Phi6 bacteriophage. The new materials were fabricated by spark plasma sintering (SPS) and/or selective laser melting (SLM) techniques and had different surface free energies and infiltration features. We show that inactivation of Phi6 in solutions with Ag and Cu ions can be as effective as inactivation by pH, temperature, or UV. Adding powder to Ag and Cu ion solutions decreased their virucidal effect. The newly fabricated ceramic and metal surfaces showed very good virucidal activity. In particular, 45%TiO₂ + 5%Ag + 45%ZrO₂ + 5%Cu, in addition to virus adhesion, showed virucidal and infiltration properties. The results indicate that more than 99.99% of viruses deposited on the new ceramic surface were inactivated or irreversibly attached to it.     

Osmoregulatory effects of prolactin and growth hormone in embryonic chicks

Intact White Leghorn chick embryos were treated daily (on Days 6-13) with bovine prolactin (PRL) or ovine growth hormone (GH) at doses of 4-10 micrograms/g embryo wet wt. A control group received an equal volume of avian saline. [Na+] and [Cl-] were determined in allantoic fluid samples taken on Days 10, 12, and 14, and in amniotic fluid and blood plasma on Day 14. Allantoic fluid, amniotic fluid, and plasma osmolarities, embryo wet weight, hematocrit, and allantoic fluid volume were also determined on Day 14. PRL-treated embryos showed significantly lower allantoic [Na+] and [Cl-] compared to controls at Days 10, 12, and 14. Allantoic fluid osmolarity was reduced, and plasma osmolarity increased, at Day 14 in PRL-treated embryos. By contrast, PRL had no effect on allantoic volume, amniotic fluid [Na+], [Cl-], or osmolarity, plasma [Na+] or [Cl-], hematocrit, or embryo wet weight. GH-treated embryos showed significantly reduced allantoic [Na+] at both Days 10 and 14, but no other treatment effect. Calculations show that the decrease in total allantoic Na+ seen in PRL-treated embryos is equivalent in magnitude to 10% of the total egg Na+. Results from studies on embryonic amphibians and mammals suggest that this sodium is likely to be sequestered in an expanded extracellular volume.     

Insulin-like growth factor-I affects amino compounds in the fluids of the chicken embryo

The concentration differences of more than 40 amino acids and related compounds in the amniotic fluid, allantoic fluid, and plasma of the chicken embryo are maintained by specific barriers. Since the amniotic and allantoic membranes are not innervated, we proposed that these barriers are controlled by hormones. Specific effects of insulin and prolactin on the amino compounds in the three fluids confirmed this hypothesis and raised the question of the possible role of growth factors. Application of insulin-like growth factor-I (IGF-I) to the chorioallantoic membrane of day 13 chicken embryos caused the following concentration changes in 41 amino compounds measured 1 and 2 h later: (1) in the amniotic fluid, an increase of 40 compounds, regardless of the presence or absence of a concomitant stress effect on these compounds; only NH3 was not affected; (2) in the allantoic fluid, a decrease of reduced glutathione (GSH) and anserine, and an increase of NH3; (3) in the plasma, a decrease of 24 compounds. Within the same time frame, stress caused in the amniotic fluid a drop of the concentration of 29, and an increase of 5, amino compounds; IGF-I reversed the stress effect on all 29 compounds the concentrations of which had dropped and enhanced the stress-induced increase of the other 5 compounds. In the allantoic fluid, stress induced an increase of GSH; IGF-I reversed this effect. In the plasma, stress caused an increase of 9 compounds; IGF-I counteracted the increase in 7 cases. These findings indicate new and unexpected roles of IGF-I in the prenatal regulation of amino compounds.     

Acute Hypoproteinemic Fluid Overload: Its Determinants, Distribution, and Treatment with Concentrated Albumin and Diuretics

Abstract. We simulated the use of massive volumes of crystalloid fluids as a treatment of acute plasma loss in a standardized experimental model and studied the factors determining the retention or excretion of the resulting acute hypoproteinemic fluid overload, its distribution within the body, and its treatment with concentrated albumin and diuretics. In accordance with the classic Starling concept, the serum protein level, i.e. the serum colloid osmotic pressure, determined the excretion/retention ratio of a given water and sodium load. Of the total fluid retention, fat and muscle each accommodated 25%, whereas the skin, which contributes only 7% to the total body weight, accounted for 37% and increased its volume by roughly one third. Concentrated albumin promoted fluid excretion in direct proportion to the achieved increment of the serum protein level and abolished the edema of fat, muscle and skin. Furosemide was virtually ineffective. The implications of these results for the 'adult respiratory distress syndrome' and disturbed wound healing are discussed and related to the concept of a critical threshold of the serum protein level.     

The avian allantois: a depot for stress-released catecholamines.

Plasma and amniotic and allantoic fluid of 10- and 14-day-old chicken embryos contain free dopamine (DA), norepinephrine (NE), and epinephrine (E). Compared with postnatal chickens, concentrations of DA and E in the plasma are very high, and they are even higher in the allantoic fluid. In contrast, the allantoic concentration of NE is below the plasma level. In the amniotic fluid, the concentrations of all three catecholamines (CAs) are below the plasma levels. High concentrations of DA and E in the allantoic fluid after opening of the egg shell decline during the following 24 hr, which indicates that they are due to stress. Asphyxia, handling, disturbance of allantoic fluid, and cooling are also perceived as stress and are followed by immediate accumulation of CAs in the allantoic fluid. DA and E respond to stress in like manner, while NE often responds with an opposite trend. It appears that the avian allantois, in addition to its role in respiration and urea disposal, also serves the instant CA removal from the circulation. Both the amniotic and the allantoic membranes of the chicken should be ideal models for the study of CA transport mechanisms.     

In vivo estrogen synthesis by the developing chicken (Gallus gallus) embryo

The concentrations of unconjugated and conjugated estrogens in the allantoic and amniotic fluids of chicken embryos have been followed during incubation. The estrogens estrone, estradiol-17β and estradiol-17α were present as their conjugates in the allantoic fluid of female embryos but not of male embryos. No estrogens were detectable in the amniotic fluid of embryos of either sex. Estradiol-17β glucuronide, the most abundant estrogen present in female allantoic fluid, was first detectable (159 pg/ml) at Stage 35 of development increasing in concentration to 4210 pg/ml at Stage 45. The concentration of estrogen in allantoic fluid of the partially decapitated embryos at Day 18 (Stage 41) of incubation was not significantly different from that of the intact stage 41, approximately Day 15 embryo.     

Disinfection efficacy and mechanism of olanexidine gluconate against norovirus

BackgroundThe purpose of this study was to evaluate the virucidal activity of a new olanexidine-containing formulation for hand hygiene (olanexidine gluconate hand rub; OLG-HR) against non-enveloped viruses and to understand its mechanism of action.MethodsThe virucidal activities of OLG-HR against two strains of caliciviruses and three adenovirus serotypes were evaluated through suspension tests. Also, virus-like particles were used to predict the effect of olanexidine gluconate on virus particle structure.ResultsThe results of suspension tests under conditions with and without interfering substances (1.5% BSA) indicated that OLG-HR had a broad-spectrum effect against non-enveloped viruses, and the virucidal effect was unaffected by organic contaminants. Furthermore, olanexidine inhibited the binding ability of virus-like particles to the binding receptor of human norovirus and increased the aggregation of virus-like particles in a dose-dependent manner. Transmission electron microscopy showed that the morphology of the virus-like particles was affected by exposure to olanexidine, indicating that the protein-denaturing effect of olanexidine gluconate caused the loss of receptor-binding capability of the viral capsid protein.ConclusionsThis study suggests that olanexidine gluconate is a potential biological and environmental disinfectant against norovirus and adenovirus.     

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.     

Ontogenesis of the pituitary-adrenal axis in the chick embryo

The concentration of corticosteroids and their metabolites in the allantoic fluid of intact and hypophysectomized chick embryos were determined for the incubation interval, 10.5 to 17.5 days. Hypophysectomy at 33–38 hr of incubation resulted in an initial reduction of allantoic fluid corticosteroids at 14.5 days of incubation; this reduction continued throughout the remainder of the incubation interval studied. Pituitary transplants, as well as ACTH administration, to hypophysectomized chick embryos restored allantoic corticosteroid concentrations to values characteristic of intact embryos. These results are considered evidence for the conclusion that in the chick embryo the pituitary-adrenal axis is established by day 14.5 of incubation.     

Pasteurized, monoclonal antibody factor VIII concentrate: establishing a new standard for purity and viral safety of plasma-derived concentrates.

A factor VIII concentrate (Monoclate-P) manufactured using a combination of pasteurization and immunoaffinity chromatography has been chosen to compare and contrast manufacturing aspects of plasma-derived factor VIII concentrates. Pasteurization is a virucidal method with a long safety record in clinical practice, while immuno-affinity chromatography selectively isolates and purifies the procoagulant protein of factor VIII, and partitions potential viral contaminants and nonessential proteins to the unbound fraction. The complete Monoclate-P production process reduces human immunodeficiency virus by > or = 10.5 log10, Sindbis (a model for hepatitis C virus) by > or = 6.5 log10, and murine encephalomyocarditis virus (a non-enveloped model virus) by 7.1 log10. The viral safety of Monoclate-P has been further demonstrated in clinical studies in patients not previously treated with blood or plasma-derived products. Additionally, the manufacture of Monoclate-P includes careful donor screening and plasma testing for antibodies to syphilis and human immunodeficiency, hepatitis B, and hepatitis C viruses to enhance source plasma safety. Combined with donor selection and plasma testing, multiple viral reduction steps effectively eliminate both lipid-enveloped viruses (e.g. human immunodeficiency, hepatitis B and C) and non-lipid-enveloped viruses (e.g. hepatitis A). In addition, polymerase chain reaction-based nucleic acid detection tests for hepatitis B and C viruses and for human immunodeficiency virus-1 have been introduced as part of an investigational new drug mechanism.     

Inactivation and Elimination of Viruses during Preparation of Human Intravenous Immunoglobulin

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.