Viruses: As mediators in "Élan vital" of the "creative" evolution.

The understanding of the processes occurring in Nature has been a continuing concern throughout the history of mankind. Intellectual tools employed towards this goal were specific for each period and have been largely based on the prevailing paradigms that reigned in the past. In this work we present evidence that supports the idea of viruses as key agents mediating natural processes linked to the evolution of organisms, particularly those involved in the flux of genes in the environment. This point of view tinges our perception of Nature and prompts us to include "viral" creativity and plasticity among the tools we employ to analyze those processes far beyond actual paradigms. Experimental data to support this proposal arose during the study of the interaction of the human pathogen, herpes simplex virus (HSV) with sulfated polysaccharides during multiplication of the virus in vitro. Sulfated polysaccharides are the main chemical structures found in carrageenans (CGNs) that are natural products obtained from seaweeds, which proved to be strong inhibitors for the virus. Here we describe the interaction between virus and CGNs as a suitable scenario for the emergence of viral variants which proved to be markedly attenuated for mice. A striking feature of these variants is that they showed changes at the level of conserved regions of the genome such as the DNA polymerase and thymidine kinase genes. In view of these findings, the importance of HSV evolution towards attenuated variants by the action of polysaccharides is also discussed. Attenuation may be considered part of a natural evolutionary process enabling the virus to contribute with valuable information for the host.

Reduced virulence of a Junin virus mutant is associated with restricted multiplication in murine cells.

C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10(2).PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.

Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice.

In the present study, the protective effect of 1T1, a lambda-carrageenan extracted from the red seaweed Gigartina skottsbergii was evaluated in a murine model of herpes simplex virus type 2 (HSV-2) genital infection. Six to eight-week-old female BALB/c mice were intravaginally inoculated with a lethal dose of HSV-2 (MS strain) and pre- or post-infection treated with different doses of a 10mg/ml solution of 1T1. A single topical administration of 1T1 shortly before infection of BALB/c mice with HSV-2 protected 9 out of 10 mice from HSV-2-induced lesions and mortality, compared with only 10% survival in control mice. In addition, 1T1 produced a total blockade in virus shedding in the vaginal secretions. When 1T1 pre-treatment was reinforced with a second dose 2h after infection, total protection was observed even when the prophylactic administration had taken place at 60min before infection. The irreversible virucidal action of 1T1 against herpes virus seems to be responsible of its protective effect against virus replication and mortality following vaginal HSV-2 infection.

[Attenuation in the virulence of a mutant of Junin virus in suckling mice]. / Atenuación en la virulencia para ratón lactante de una mutante de virus Junín.

The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.

Virus driven evolution: a probable explanation for "Similia Similibus Curantur" philosophy.

Despite the advances in biomedical knowledge, there remain many challenging and significant unsolved problems among which are included viral pathogenesis and antiviral therapy, as main topics in human health. On this respect, for instance, our knowledge about human immunodeficiency virus and AIDS is still insufficient to deal with problems of immense significance, such as the possible "natural cure" for a chronic infection or the induction of protective immunity against this agent. At the same time, new viral diseases of humans and animals continue to emerge or re-emerge, due to changes in host susceptibility and/or in virus virulence as well as to re-introduction of a virus that had disappeared from a defined population. These changes, at least in part, may appear as a consequence of antiviral therapies and lead to the selection of viral mutants. Moreover, taking into account that viruses have been studied as causative agents of conspicuous diseases a broad spectrum of uncertainty is still present when unapparent persistent infections are considered. Based on Hippocrates (460-357 b.C.E) natural philosophy, "Natura Morborum Medicatrix" which represents the natural healing force, i.e.: "Nature cures diseases"; and "Similia Similibus Curantur" which means "like cure like", we propose the use of natural compounds with chemical structures similar to cellular membrane components. On this approach, sulfated polysaccharides obtained from marine algae may act as a driving force for the emergence of attenuated viruses, enabling this way a practical approach for preventive therapies for herpes simplex virus infection. At the same time, viruses would be creative tools and their contribution by adding new genetic identity to their host are set points of genesis in the growth of the tree of life.

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro.

In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.

Antiviral activity of a carrageenan from Gigartina skottsbergii against intraperitoneal murine herpes simplex virus infection.

The partially cyclized mu/nu-carrageenan 1C3, isolated from the red seaweed Gigartina skottsbergii, was previously shown to be a potent inhibitor of the in vitro replication of Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here the protective effect of 1C3 in a murine model of intraperitoneal ( i. p.) HSV-1 infection was evaluated. OF1 mice were i. p. infected with 5 x 10 (5) PFU of HSV-1 KOS strain, and the effects of different treatments with 1C3 were studied. When 30 mg/kg of body weight of 1C3 was administered by the i. p. route immediately after HSV-1 infection, 87.5 % survival of the animals was achieved (p < 0.005), associated with a delay in the mean day of death in 1C3-treated non-surviving mice. Animal survival was not improved when multiple doses of 1C3 were also given in the period 1 - 48 h post-infection, and no protection was afforded when treatment was started after 24 h of infection. When virus and compound were injected by different routes, i. p. and intravenous ( i. v.), respectively, a still significant protection was achieved (40 % survival, p < 0.05). No toxicity of 1C3 for the animals was recorded. The pharmacokinetic properties were analyzed after injection of 1C3 into the tail vein by monitoring of [ (3)H]-1C3 in plasma and organs and by a bioassay of the anti-HSV-1 activity remaining in serum after non-radioactive 1C3 inoculation. A very rapid disappearance of the compound from the blood was observed since only 5.9 - 0.9 % of the radioactivity of the initially administered [ (3)H]-1C3 appeared in the plasma between 5-300 minutes after administration. A transient peak of radioactivity was detected in the kidney 15 minutes after inoculation. The bioassay confirms the presence of the compound circulating in a biologically active form up to 1 hour after injection.

Calomys musculinus, the natural reservoir of Argentine haemorrhagic fever, is a semipermissive host for a mouse-attenuated mutant of Junin virus.

C167, a mouse-attenuated strain of Junin virus derived from the XJC13 strain, also displayed reduced virulence for the South American cricetid Calomys musculinus, a natural reservoir of this virus in nature. Intracerebral inoculation of C. musculinus with 500 PFU of C167 produced only 25% mortality, whereas the parental XJC13 killed 85% of the animals. The attenuation of C167 for this cricetid was lower than for albino mice. The multiplication of C167 in C. musculinus-derived embryo or kidney fibroblasts was diminished with respect to XJC13, allowing us to define C. musculinus cells as a semipermissive system for C167, whereas murine and Vero cells were restrictive and permissive cultures, respectively. As a consequence, C167 as well as XJC13 were able to establish a persistent infection in C. musculinus embryo fibroblasts and Vero cells, but the mutant could not induce a carrier state in murine cells. Thus, the degree of susceptibility of C. musculinus to C167 was linked to the semipermissiveness of cricetid cells to virus multiplication.

Glycoprotein-mediated biological properties of a host range mutant of Junin virus.

The biological properties mediated by the main envelope glycoprotein (GP1) of a mouse-attenuated mutant of Junin virus, named Cl67, were investigated in comparison with its parental strain XJCl3. In contrast to the parental strain, this mutant was unable to multiply in primary cultures of mouse embryo fibroblasts (MEFs). Impairment of Cl67 multiplication was associated with a lack of virus binding to MEF, probably due to an altered interaction between GP1 and cellular receptors. Antigenic and immunogenic characterization of GP1, performed by neutralization assays, demonstrated that, under certain conditions, polyclonal and monoclonal antibodies exhibited differential affinity and specificity for each virus. Cl67-infected Vero cells showed a marked pH-dependent fusion capability, suggesting more efficient low pH triggering of fusion by mutant virus GP1 in comparison with the parental strain.

Experimental infection of suckling mice with a host range mutant of Junin virus.

Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.

A mouse attenuated mutant of Junin virus with an altered envelope glycoprotein.

The XJC13 strain of Junin virus (JV) and the mouse-attenuated mutant C167 showed different GP38 peptide mapping after limited proteolysis with ficin and papain; viral infectivity of both viruses also exhibited a different susceptibility to protease treatment. A correlation between envelope glycoprotein alteration and JV virulence in neonatal mice is proposed.

Herpes simplex virus type 1 variants arising after selection with an antiviral carrageenan: lack of correlation between drug susceptibility and syn phenotype.

Natural carrageenans of diverse structural types isolated from the red seaweed Gigartina skottsbergii were recently identified as potent and selective inhibitors of herpes simplex virus types 1 and 2 (HSV-1 and -2). The mu/nu-carrageenan 1C3 was tested in vitro for its ability to select resistant variants. After serial passages of HSV-1 strain F in Vero cells in the presence of increasing concentrations of 1C3, viruses emerged that were approximately 2- to 10-fold more resistant to 1C3 inhibition than parental virus; these viruses formed large plaques with an altered syncytial phenotype (1C3-syn). Plaque-purified syncytial variants isolated from passages 13 and 14 have shown variable levels of resistance to 1C3, as well as to the other antiviral carrageenans isolated from G. skottsbergii and to other sulfated polysaccharides with known antiviral activity, such as heparin and dextran sulfate 8000, but all the clones were susceptible to acyclovir. The syn phenotype was not related to polysaccharide resistance. All the 1C3-syn variants formed large syncytia in Vero and CV-1 cells but did not induce fusion in other cell types. The growth efficiency in Vero cells, as well as the virulence for mice by intracerebral or intraperitoneal inoculation of 1C3-syn variants, showed no significant alterations in comparison with the parental virus. The syncytial properties were not affected by cyclosporine or melittin, suggesting that an alteration on glycoprotein gB could be responsible for the syn phenotype induced by 1C3.

Inhibitory action of natural carrageenans on Herpes simplex virus infection of mouse astrocytes.

The effects of the carrageenans 1T1 (lambda-type), 1C1 (kappa/iota-type) and 1C3 (upsilon/nu-type), isolated from the red seaweed Gigartina skottsbergii, on herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection of murine astrocytes were investigated. The three compounds were effective inhibitors of virus replication, as determined by a virus yield inhibition assay, in astrocytes as well as in Vero cells. The 50% inhibitory concentration was in the range of 0.9-3.6 and 0.4-3.2 microg/ml for astrocytes and Vero cells, respectively, whereas the 90% inhibitory concentration ranged from 6.5 to 17.0 microg/ml in astrocytes and from 3.5 to 22.2 microg/ml in Vero cells. No cytotoxicity was detected at concentrations of up to 1,000 microg/ml, indicating high selectivity indices for these compounds. Inhibition of viral cytopathology and antigen expression was also detected in the presence of the carrageenans. The increase in the expression of glial fibrillary acidic protein (GFAP), an activated astrocyte marker, produced during the course of HSV-1 infection in astrocytes, was reversed in the presence of 1C1 and 1C3. By contrast, the lambda-carrageenan 1T1 increased the expression of GFAP, independently of HSV-1 infection.

A comparison of Junin virus strains: growth characteristics, cytopathogenicity and viral polypeptides.

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.

Antiviral activity of natural sulphated galactans on herpes virus multiplication in cell culture.

A sulphated galactan (SG) with low molecular weight (app. 2800) was isolated from extracts of Cryptopleura ramosa, a red seaweed from the South American coasts. The compound was a selective inhibitor of HSV-1 and HSV-2 replication in Vero cells with 50% inhibitory concentrations (IC50) in the range 1.6-4.2 micrograms/ml and a 50% cytotoxic concentration (CC50) of 476 micrograms/ml. SG was also effective against HSV-1 in cells of neural origin such as murine astrocytes. The mode of action of SG could be ascribed to an inhibitory action on virus adsorption. Furthermore, SG did not inhibit the blood coagulation process at concentrations highly exceeding the IC50.

Atenuación en la virulencia para ratón lactante de una mutante de virus Junín. / [Attenuation in the virulence of a mutant of Junin virus in suckling mice]

The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50

lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.