Large-scale purification of high purity α1-antitrypsin from Cohn Fraction IV with virus inactivation by solvent/detergent and dry-heat treatment.

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Antiviral activity of a novel composition of peracetic acid disinfectant on parvoviruses.

Porcine parvoviruses (PPV) are known to be particularly resistant to many disinfectants used to control other non-enveloped viruses. However, effective disinfectants used against PPV are harsh and corrosive to animal health facilities and the environment. We propose a noncorrosive "green" disinfectant that generates peracetic acid in-situ and is capable of inactivating PPV completely at a 1% concentration for a 10-minute contact time.

Les parvovirus porcins (PVP) sont reconnus pour être particulièrement résistants à plusieurs désinfectants utilisés pour éliminer d'autres virus non-enveloppés. Toutefois, les désinfectants efficaces utilisés contre le PVP sont rudes et corrosifs pour les installations de santé animale et l'environnement. Nous proposons un désinfectant «vert¼ et non-corrosif qui génère de l'acide peracétique in situ et qui est capable d'inactiver le PVP complètement lorsqu'utilisé à une concentration de 1 % pour un temps de contact de 10 minutes.(Traduit par Docteur Serge Messier).

Bioactive isoquinoline alkaloids from Corydalis saxicola.

Twelve isoquinoline alkaloids including two new nitro-containing tetrahydroprotoberberines, (-)-2,9-dihydroxyl-3,11-dimethoxy-1,10-dinitrotetrahydroprotoberberine (1) and (+)-4-nitroisoapocavidine (2), were isolated from the whole plant of Corydalis saxicola Bunting. The structures of the new compounds were established by spectroscopic analysis and chemical evidence. The inhibitory activity of these isolates against cholinesterase and canine parvovirus were evaluated. Compounds 1 and 1A, (+)-1-nitroapocavidine (5), berberine (8), palmatine (9), dehydrocavidine (10), and sanguinarine (11) showed potent inhibitory activity against acetylcholinesterase with IC(50) values of less than 10 µM, while only compound 1 possessed weak activity against canine parvovirus. Structure-activity studies demonstrated that the nitro substituents at ring A in the tetrahydroprotoberberines led to an increase in the anti-acetylcholinesterase activity.

Suppression of proliferation of poliovirus and porcine parvovirus by novel phenoxazines, 2-amino-4,4 alpha-dihydro-4 alpha-7-dimethyl-3H-phenoxazine-3-one and 3-amino-1,4 alpha-dihydro-4 alpha-8-dimethyl-2H-phenoxazine-2-one.

The present study aimed at investigating the antiviral effects of 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2) on 6 representative viruses: poliovirus, porcine parvovirus, simian virus 40 (SV-40), herpes simplex virus-1 (HSV-1), Sindbis virus, and vesicular stomatitis virus (VSV). Phx-1 and Phx-2 suppressed the proliferation of poliovirus in Vero cells and that of porcine parvovirus in ESK cells at concentrations between 0.25 microg/ml and 2 microg/ml, when the cells were treated with Phx-1 and Phx-2 for 1 h and then inoculated with these viruses. The proliferation of the other viruses, SV-40, HSV-1, Sindbis virus, and VSV, in the host cells was not influenced by Phx-1 or Phx-2 at concentrations less than 20 microg/ml. The results suggest that Phx-1 and Phx-2 may be useful to prevent the proliferation of poliovirus and porcine parvovirus infection and may contribute to developing new antiviral drugs in future.

[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.

Infection of apheresis cells by parvovirus B19.

Parvovirus B19 is the only member of the Parvoviridae family known to cause disease in humans. Owing to the high level of cell tropism the virus can only replicate in proliferating and differentiating erythroid precursor cells, which are present in human bone marrow and foetal liver. As human bone marrow is very difficult to obtain, an alternative in vitro system for the propagation of B19 virus has been developed, based on the application of mobilized haemapoietic progenitor (apheresis) cells. These cells are routinely harvested from cancer patients after treatment with recombinant human granulocyte/macrophage colony-stimulating factor. Replication of parvovirus B19 in vitro is possible in these cells after stimulation with erythropoietin. Therefore, this system is an easily, accessible alternative to the use of human bone marrow in parvovirus B19 infection assays.

Water-insoluble camptothecin analogues as potential antiviral drugs.

In addition to being causative agents of infectious diseases in animals and humans, DNA viruses have served as models for the study of eukaryotic molecular mechanisms including replication and transcription. Studies of DNA virus functions utilizing cell-free systems and virus-infected cells in culture, in the presence of the anticancer drug camptothecin (CPT), have demonstrated that CPT is a potent inhibitor of replication, transcription and packaging of double-stranded DNA-containing adenoviruses, papovaviruses and herpesviruses, and the single- stranded DNA-containing autonomous parvoviruses. CPT inhibits viral functions by inhibiting topoisomer- ase I, a host cell enzyme required for initiation and completion of the viral functions. These findings indicate that CPT analogues could be developed for use as potent drugs against DNA viruses.

Antiviral activity of some Nigerian medicinal plant extracts.

Plants from Northern Nigeria with a history of use in both human and veterinary traditional medicine have been investigated for their antiviral activity and their cytotoxicity determined. Extracts were tested against poliovirus, astrovirus, herpes simplex viruses and parvovirus, using the microtitre plate inhibition tests. Most of the extracts have activity against more than one virus at a dose rate of between 100 and 400 microg/100 microl.

Comparison of two different methods for inactivation of viruses in serum.

In order to compare protocols for inactivation of viruses potentially present in biological specimens, three different model viruses were treated in bovine serum by two different inactivation methods: samples were subjected either to chemical inactivation with ethylenimine (El) at concentrations of 5 and 10 mM at 37 degrees C for periods up to 72 h or to electron-beam irradiation in frozen and liquid form with doses varying between 11 and 46 kGy. The chemical inactivation resulted in nonlinear tailing curves in a semilogarithmic plot of virus titer versus inactivation time showing non-first-order kinetics with respect to virus titer. The time for inactivation of 7 log10 units of porcine parvovirus (PPV) was about 24 h for both El concentrations, whereas 5 log10 units of bovine viral diarrhea virus (BVDV) was inactivated in 2 h for both El concentrations and 6 log10 units of porcine enterovirus (PEV) was inactivated within 3 h. The inactivation with electron-beam irradiation resulted in almost linear curves in a semilogarithmic plot of virus titer versus irradiation dose, reflecting a first-order inactivation. The rate of inactivation was almost twice as fast in the liquid samples compared to the rate in frozen ones, giving values of the doses needed to reduce virus infectivity 1 log10 unit for inactivation of PPV of 11.8 and 7.7 kGy for frozen and liquid samples, respectively, whereas the corresponding values for BVDV were 4.9 and 2.5 kGy, respectively, and those for PEV were 6.4 and 4.4 kGy, respectively. The nonlinear inactivation with El makes it impossible to extrapolate the curves beyond the virus detection limit and thereby predict the necessary time for complete inactivation, i.e., to a level beyond the detection limit, of virus in a given sample. The first-order inactivation obtained with electron-beam irradiation makes such a prediction possible and justifiable. The two methods are discussed with respect to their different kinetics and applicability under different circumstances and criteria for inactivation, and considerations for choice of method are discussed.

Ozone disinfection dynamics of enteric viruses provide evidence that infectious titer reduction is triggered by alterations to viral colloidal properties.

The inactivation dynamics of three enteric virus species (polio-, rota- and parvovirus) were analysed in different aqueous suspensions by using O3 under continuous flow conditions. A mathematical model for the reaction rate of infectious titer reduction was proposed, based on the thermodynamic principles of phase behaviour of colloids suspended in aqueous environments. Up to a certain threshold dosage of residual ozone (RO), and depending on the type of test virus and the ionic or organic load in the stock suspension, the logarithm of the reaction rate constant of viral inactivation rate was observed to vary in a rather sigmoidal manner with log RO concentration. Data from photon correlation spectroscopy, electron microscopy and tensiometric analysis suggested that below the threshold RO, the pattern of virus inactivation dynamics reflects the varying potential of different-sized viral particles (VPs) to adsorb to the cellular monolayer. There is strong evidence that oxidant-induced surface activity of organic matter causes redistribution of VP infectivity. This hypothesis was statistically corroborated inasmuch as experimental inactivation data proved to be satisfactorily fitted by a logistic equation. It was concluded that viral infection, and thus viral inactivation, is a complex process which is governed largely by the classical laws of colloidal behaviour. The latter is suggested to appreciably determine the capability of inoculated VPs to infect host cultures. This notion may especially be cause for concern when regulatory requirements for virus disinfection are being based on titration results from in vitro testing procedures.