Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue

Recently reported qualitative experiments proved that retroviral infectivity is not destroyed by lyophilization performed on systemically infected bone and tendon. The now accomplished quantitative determination of residual infectivity for enveloped and non-enveloped viruses allows a validation of the production process regarding viral safety in freeze-dried bone transplants. The lyophilization effect on the infectivity of two non-enveloped viruses (Maus Elberfeld virus, MEV; Porcine parvovirus, PPV) and one enveloped virus (Vesicular Stomatitis virus, VSV) was examined for virus-spiked bone material in comparison to lyophilized viruses, original virus stock, and air-dried viruses. All experiments were carried out with both cell-free and cell-associated virus. Significant differences were observed regarding the reduction of virus titers (TCID50). Infectivity of VSV was reduced by about 3-4 log10 using lyophilization in presence of bone matrix and of MEV by 6-7 log10, while no substantial reduction in virus titers was observed for PPV. Lyophilization of cell-free or cell-associated virus is not sufficient to inactivate viruses completely. However, lyophilization could have an additive effect in line with other production steps used in the manufacturing process.     

High specific infectivity avian RNA tumor viruses

Conditions for obtaining avian RNA tumor viruses of high specific infectivity were investigated. The following results were obtained:

1. (1) Pr-C-RSV showed highest specific infectivity when harvested from virus-producing chick embryo fibroblasts at 20-min intervals. Such virus was 5 times more infectious than virus harvested at 30-sec intervals, and 10 times more infectious than virus harvested at 24-hr intervals. Other cloned nondefective RNA tumor viruses showed similar patterns.

2. (2) Prompt chilling of virus collected at frequent intervals was essential because otherwise it was heat-inactivated rapidly. Virus harvested at 24-hr intervals was heat-inactivated much more slowly. However, this difference was not due to differences in the virus particles, but to differences in media that had been in contact with cells for short or long periods of time, respectively.

     

Effect of sucrose phosphate and sorbitol on infectivity of enveloped viruses during storage.

Cytomegalovirus and varicella-zoster virus recovery from sucrose phosphate (0.2 M SP) and 70% sorbitol (sorbitol) was compared after storage at -70, 4, and 20 degrees C over time. Recovery from 0.2 M SP was uniformly better. More tissue culture infective doses and infectious foci were recovered in cell monolayers inoculated with 0.2 M SP virus stocks as compared with viruses stored in 70% sorbitol. Although both viruses were isolated from diluted fresh stocks (10(-1) through 10(-4], freezing diluted virus suspensions generally resulted in diminished recovery. Similar stabilizing effects on respiratory syncytial virus and herpes simplex virus type 1 infectivities were observed when stocks were preserved in 0.2 M SP, as compared with 70% sorbitol. Overall, 0.2 M SP was better than 70% sorbitol for stabilizing cytomegalovirus, varicella-zoster virus, respiratory syncytial virus, and herpes simplex virus type 1 infectivities under the conditions tested.     

Rafts, anchors and viruses--a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors

Lipid rafts have been proposed as sites for the assembly of a number of viruses and are considered to play a major role in pseudotyping events. As a consequence, host glycosylphosphatidylinositol (GPI) anchored proteins commonly associated with lipid rafts can be found being incorporated into viral lipid envelopes with beneficial consequences for viral replication. In this review we will look at the link between lipid rafts, GPI-anchored proteins and retroviral particles and how these relationships can be exploited for the modification of enveloped viruses.     

Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses

The covalent attachment of fatty acids to the glycoproteins of orthomyxo-, paramyxo, alpha-, and coronavirus was studied. All enveloped viruses analyzed afford covalently bound fatty acid in at least one species of their spike glycoproteins. No internal components of the viruses studied including the hydrophobic M proteins of myxo- and rhabdoviruses contained fatty acid. Analysis of myxovirus particles devoid of the exposed portions of their spikes revealed that fatty acids are linked to the hydrophobic tail fragment of the glycoprotein which is associated with the viral lipid bilayer. With influenza virus hemagglutinin the fatty acid attachment site could be located at the cyanogen bromide peptide of the small subunit (HA₂) which contains the membrane-embedded region of the polypeptide. The binding of fatty acids to viral glycoproteins occurs in a wide range of host cells including mammalian, avian, and insect cells.     

On the use of chloramine-T to iodinate specifically the surface proteins of intact enveloped viruses.

Rous-associated virus-6I was used as a model for studying the labelling specificity of the chloramine-T iodination procedure when applied to intact enveloped viruses. The specificity of labelling depended markedly on the concentration of iodide in the reaction mixture. At low concentrations of iodide (below 0-5 muM) only the surface proteins and lipid of intact virions were iodinated; there was no detectable labelling of internal proteins. At 10 muM-iodide, however, both internal and external proteins were iodinated; moreover there was a marked change in the reactivity of the surface proteins. It appears that the lipid envelope provides an effective barrier to the iodinating complex generated at low, but not at high, concentrations of iodide. These and other observations suggest that the chloramine-T procedure has a previously unrecognized potential for specifically labelling the surface proteins of lipid-enveloped structures.     

Human cancers and viruses: a hypothesis for immune destruction of tumours caused by certain enveloped viruses using modified viral antigens.

Certain viruses which have been identified as possible aetiological agents of human malignant tumours have 2 common characteristics: a) they persist in the human body for long periods despite the presence of antibodies to them and b) they all possess viral envelopes. The envelopes, consisting of phospho-lipoproteins are derived from host cells viz nuclear envelope in the case of DNA viruses, and the cell membrane in the case of RNA viruses. These host cell elements on the viral envelope modify the antigenicity of the specific surface antigens which are now perceived by the host immune system as partly self. This in turn blackmails the immune system, if it is to avoid serious auto-immune disease, into producing compromise and ineffective antibodies. The hypothesis proposes the dissolution of the viral envelope in vitro and the re-introduction of the viral core into the host. This should provoke a new uncompromised immune response because it will be directed at the viral core only. This response should eliminate the viral core and with it, the whole enveloped virus, as well as the malignant tumour cells which carry the viral genome derived essentially from the viral core. This approach should introduce a new method for treating and preventing tumours caused by enveloped viruses and the chronic diseases caused by such viruses.     

Current status on the development of pseudoviruses for enveloped viruses

Emerging and reemerging infectious diseases have a strong negative impact on public health. However, because many of these pathogens must be handled in biosafety level, 3 or 4 containment laboratories, research and development of antivirals or vaccines against these diseases are often impeded. Alternative approaches to address this issue have been vigorously pursued, particularly the use of pseudoviruses in place of wild‐type viruses. As pseudoviruses have been deprived of certain gene sequences of the virulent virus, they can be handled in biosafety level 2 laboratories. Importantly, the envelopes of these viral particles may have similar conformational structures to those of the wild‐type viruses, making it feasible to conduct mechanistic investigation on viral entry and to evaluate potential neutralizing antibodies. However, a variety of challenging issues remain, including the production of a sufficient pseudovirus yield and the inability to produce an appropriate pseudotype of certain viruses. This review discusses current progress in the development of pseudoviruses and dissects the factors that contribute to low viral yields.     

Coat protein is required for infectivity of tobacco streak virus: biological equivalence of the coat proteins of tobacco streak and alfalfa mosaic viruses.

The four nucleoprotein components of tobacco streak virus (TSV) were purified by zonal centrifugation. Each component contains mainly one RNA species. Infectivity studies indicate that the three fastest-sedimenting nucleoproteins are required for viral replication. A mixture of the three largest RNA's however, is not infectious but can be activated by the viral coat protein or by the smallest TSV-RNA. The same situation exists in alfalfa mosaic virus (AMV). The coat proteins of TSV and AMV, which are rather different as judged by serology and tryptic digestion products, cannot only activate their own genome but also that of each other. The nucleoprotein components of the two viruses, however, could not be substituted for each other.TSV-RNA is as efficient as AMV-RNA in withdrawing protein subunits from AMV nucleoprotein. However, TSV nucleoprotein could not be uncoated by its own RNA or by AMV-RNA. Nevertheless, the affinity of TSV-RNA for AMV coat protein and the functional equivalence of the two coat proteins point to a possible common origin of these two viruses.     

Inhibition of influenza viral neuraminidase activity by collectins

Summary The collectins, lung surfactant proteins A and D (SP-A and SP-D), contribute to innate host defense against influenza A virus (IAV) in vivo. Although collectins bind to the viral hemagglutinin (HA) and inhibit early stages of viral infection in vitro, they also bind to the neuraminidase (NA) and inhibit NA activity. We used a variety of NA functional assays, viral strains and recombinant (mutant or wild type) collectins to characterize the mechanism of NA inhibition. NA inhibition by SP-D correlates with binding of its carbohydrate recognition domain (CRD) to oligomannose oligosaccharides on the viral hemagglutinin (HA). The effects of SP-D are additive with oseltamivir, consistent with differences in mechanism of action. NA inhibition was observed using fetuin or MDCK cells as a substrate, but not in assays using a soluble sialic acid analogue. Collectin multimerization and CRD binding properties are key determinants for NA inhibition. SP-D had greater NA inhibitory activity than mannose-binding lectin, which in turn had greater activity than SP-A. The markedly greater NA inhibitory activity of SP-D compared to SP-A may partly account for the finding that deletion of the SP-D gene in mice has a greater effect on viral replication in vivo.     

Inhibition of avian sarcoma virus replication by glucosamine: a specific effect on the synthesis and processing of viral proteins.

Treatment of avian RNA tumor virus-infected chicken fibroblasts with 20 mM glucosamine for 8 hr leads to the inhibition of virion synthesis (Hunter et al., 1974). In this report we have investigated the mechanism of inhibition. A kinetic analysis on the inhibition of virion production revealed that the effect of glucosamine was essentially complete 5 hr after its addition, by this time virion production has been inhibited 50-fold. An analysis of the effect of glucosamine on cellular and intracellular virus protein synthesis revealed that by 5 hr cellular protein synthesis was inhibited 60% and virus protein synthesis was specifically inhibited by a further 75%. The major nonglycosylated viral structural proteins (p27, p19, p15, and p12) are synthesized as a 76,000 dalton precursor (Pr76) which is subsequently cleaved to the virion proteins. The effects of glucosamine on this process were examined by pulse-chase experiments. After 5 hr of treatment with glucosamine only 5% of the precursor synthesized in a 15-min pulse was cleaved in an hour, compared with 54% in untreated cells. Chases in the absence of glucosamine demonstrated that the precursor synthesized in the presence of glucosamine could be cleaved and assembled into virions. SDS-PAGE analysis of these virions showed that they lacked radioactively labeled glycoproteins, gp85 and gp37. Thus, glucosamine inhibits the replication of avian RNA tumor viruses by a combination of an inhibition of cellular protein synthesis, viral structural protein synthesis, and Pr76 cleavage.     

Inhibition of HIV and SIV infectivity by blockade of alpha-glucosidase activity.

Processing of HIV and SIV envelope oligosaccharides is critical for proper intracellular trafficking and function. An inhibitor of alpha-glucosidases I and II, N-butyl deoxynojirimycin (N-BuDNJ), retards HIV-1 and SIVmac spread in lymphocytes and monocytes by diminishing virus infectivity, and also causes a reduction in syncytia formation between infected cells and uninfected lymphocytes. N-BuDNJ retards envelope processing from the precursor form to the mature surface (SU) and transmembrane proteins in HIV-1- and SIVmac-infected cells, as well as in cells infected with vaccinia-HIV-1 envelope recombinant virus. However, no significant reduction is seen in the amount of SU in released virus particles, though the virus particle-associated SU from N-BuDNJ-treated cells has an altered electrophoretic mobility. In contrast, N-BuDNJ had no effect on GAG protein synthesis and processing. These findings demonstrate a critical requirement for oligosaccharide processing by alpha-glucosidases I and II for HIV-1 and SIVmac envelope processing and fusogenicity.     

Inhibition of MHC class I antigen presentation by viral proteins

 An essential part of the immune response to viral infections is the recognition and elimination of infected cells by cytotoxic T lymphocytes. For this purpose a display mechanism has evolved which is present in almost all nucleated cells: the major histocompatibility complex class I antigen processing pathway. Both self and foreign antigens are degraded in the cytosol to peptides which are translocated into the endoplasmic reticulum where they are loaded onto MHC class I molecules. Pathogens living inside the cell are evolving under the constant selection pressure of such immune surveillance. As a result such infectious organisms have developed a variety of strategies to prevent their antigens from being presented. Since our understanding of the cell biology of antigen presentation has greatly advanced in recent years, it has now become possible to unravel several of the molecular mechanisms by which viruses interfere with MHC class I antigen presentation. Examples for the interference of viral molecules with components of the MHC class I pathway are presented in this review. Received: 12 June 1996 / Accepted: 24 July 1996     

Enhancement of viral infectivity by cytochalasin

Cytochalasins B and D enhance the infectivity of poliovirus; a 5-fold increase in viral titer occurs on treatment of cells with 4–6 μg/ml of either congener. At lower concentrations, however, CD is more effective than CB in increasing the infectibility of cells. The enhancement of viral infectivity occurs whether CD is applied to the cells before and during viral attachment, or after penetration has occurred. CD does not affect the attachment, penetration, or elution of radiolabeled poliovirus, and only minimally stimulates uncoating. Enhancement of infectivity progressively declines as the time of addition of cytochalasin after viral penetration is prolonged. We conclude that cytochalasin affects some event(s) early in the eclipse period of polioviral development. Pretreatment of Vero cells with dibutyryl cyclic adenosine 3′:5′-monophosphate antagonizes the effect of CD on the infectivity of poliovirus.Cytochalasin also increased the infectivity of other RNA viruses including parainfluenza virus. However, no augmentation of infectivity is found with vaccinia, and infectibility with adenovirus is decreased by CD.     

Identification of herpes simplex viruses by automated profile analysis of viral proteins

In this report we describe a computerized virus identification system based upon the fact that viral proteins, separated by one-dimensional (ID) SDS-PAGE have unique banding patterns (protein profiles). Fifty-eight clinical strains of herpes simplex virus (HSV) were correctly identified by comparing their protein profiles with reference profiles of HSV, other viruses and uninfected cell cultures stored in a computer data base.     

Cell culture density modulates the incorporation of HLA antigens by enveloped viruses

Uninfected, as well as feline leukemia virus (FeLV) infected human cells cultured under high cell density conditions undergo changes in the expression of major histocompatibility complex (MHC) antigens, as determined by indirect trace binding radio immunoassay (RIA) using monoclonal anti-HLA antibodies and by decreased sensitivity to complement mediated cytotoxicity by anti-HLA alloantibodies. FeLV particles produced by the viral infected cells are also sensitive to neutralization by anti-HLA antibodies, suggesting that enveloped viral particles incorporated MHC antigens in the viral envelope. The amount of HLA antigens expressed in the viral enveloped, closely reflects the expression of HLA antigens by the virus-producer lymphoid cells. FeLV-infected HsB-2 (T) and SB (B) lymphoid cells cultured under high cell concentration condition show decreased expression of some HLA antigens (A2, B12, B17), and the viral particles produced by those cells also incorporate lower amounts of such antigens. Our results, based on the findings that human lymphoid cells (uninfected, as well as FeLV infected) show decreased expression of some HLA membrane determinants when growth under high cell density conditions, indicate that no viral selective mechanism operates in the incorporation of HLA determinants by enveloped viruses. Instead, our results suggest that viruses pick up MHC antigens from the host cell membrane according to the concentration of those antigens on the surface of the cells at the time of virus budding.