Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis.

Exposure of influenza virus to an acidic environment, which is known to be required for viral fusion and hemolysis, has recently been shown to induce a conformational change in the hemagglutinin molecule. In the present study, we examined the effects of acid incubation on the antigenicity, biological activity, and morphology of influenza virus A/PR/8/34 (H1N1). Incubation of PR8 virus at pH 5 in the absence of erythrocytes resulted in a rapid and irreversible loss of viral hemolytic activity and infectivity. Apart from a less distinct appearance of the viral surface projections and slight damage to the envelope structure, acid incubation did not result in gross morphological changes in the viral architecture. The acid-induced change could be detected in the form of greatly increased or decreased binding of many monoclonal antibodies directed to each of the four major antigenic regions of the hemagglutinin. Triggering of viral hemolytic activity and antigenic alterations was similarly pH dependent. In addition, the different pH dependencies of egg-grown and trypsin-treated MDCK-grown viruses coincided with an analogous pH dependence of the antigenic alterations that were observed with these viruses. These observations are compatible with the idea that some of the anti-hemagglutinin antibodies detect conformational changes in the hemagglutinin which are required for the initiation of fusion and hemolysis.     

Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp¹⁵³ and Glu¹⁵⁸, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His¹⁴⁸ was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different pH values.

Vesicular stomatitis virus (VSV)-mediated cell fusion from without can be induced by transient exposure to low pH, subsequent to adsorption of VSV at neutral pH. To study the mechanism of VSV-induced cell fusion, we examined the effect of pH condition at virus adsorption on acid-inducible VSV-mediated cell fusion. Although the binding of VSV to BHK-21 cells was most efficient under acidic condition (pH 5.7-6.3), extensive cell fusion was not observed under this condition. A temporary exposure to low pH after binding at neutral pH also decreased fusion activity. However, return to neutral pH for 2 min just after the acid binding restored the fusion activity. These results indicate the requirement of neutral pH condition for VSV-mediated cell fusion prior to the acid stimulation which induces conformational change of the virus glycoprotein into a fusogenic form.     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.     

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

FUSION OF INTACT HUMAN ERYTHROCYTES AND ERYTHROCYTE GHOSTS

Sendai virus is able to induce the fusion of human erythrocytes. Bivalent cations or ATP are not essential for polyerythrocyte formation. High fusion indices were obtained when Sendai virus was added to cells incubated in the presence of both EDTA and iodoacetic acid. Human erythrocyte ghosts prepared by gradual hemolysis still retain the potential to undergo virus-induced fusion. Fusion of human red blood cells without the addition of viruses was obtained by incubation of erythrocytes at pH 10.5 in the presence of Ca⁺⁺ (40 mM) or by addition of phospholipase C Clostridium perfringens preparations to cells previously agglutinated or polylysine.     

Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.     

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.