**4. Viruses: Friend or enemy**

It is tempting to postulate that the driving forces of evolutionary novelty are not randomly derived from chance mutations of the genetic text, but from a precise genome editing by omnipresent viruses [58]. For decades, non-coding regions of the genome have been ignored or declared as "junk"-DNA. Recently, scientists have realized that these regions incorporate decisive higher-order regulatory functions. New research has shown that these non-coding repetitive sequences originated primarily from retroviral RNA [59,60].

For a long time, viruses were interpreted as causing acute infections of susceptible organisms, using the host cellular machinery to reproduce, and achieving their lytic nature only in order to infect other cells. Although this narrative remains valid, it merely represents a case of viruses that were unable to reach persistent or chronic status of infection [61]. Most viruses, however, are stable, persistent agents that are able to establish a complex relationship with the host cell and in many cases this interaction lasts for the entire lifespan of the cell even when a competent immune system is present. The immune system is of crucial importance in defense against infection. It has to cope with a large number of different pathogens that relentlessly develop new ways to avoid recognition or elimination. Yet most infections are cleared. Immune-system genes must evolve to keep pace with increasingly sophisticated evasion by pathogens. To remain effective, defense demands creativity and competence because many pathogens have sophisticated and rapidly evolving evasion mechanisms [62]. This defense response is triggered by the presence of pathogens within the host, however there exists an immune response that comprises a set of autoreactive "natural antibodies" that do not rely on exogenous antigen stimulation to be synthesized by autoantibody-secreting B lymphocytes [63,64]. On the other hand, these autoreactive natural antibodies are reactive against components of the host´s immune system (i.e. cytokines) exacerbating ongoing infectious diseases or predisposing host to infection [65,66].

Individual resistance to pathogens depends on the combination of receptors on cells from the immune system although non-immune genes also influence resistance [67]. Signs of natural selection in a human population are especially illustrative, when a mutation in a certain gene is dangerous in normal conditions but confers resistance to infections widespread in the region. Among the better known are the mutations in hemoglobin and glucose-6-phosphate-dehydrogenase affecting red blood cells and conferring resistance to malaria [68]. Another example is the deletion at the 5´end of the CCR5 chemokine receptor conferring resistance to HIV infection. This molecule serves as the principal co-receptor, with CD4, for HIV-type 1. The allele with the deletion was intensely selected in Europe probably because it also provided resistance to plague and smallpox [69]. More subtle, but nonetheless important, relationship between cell and virus is that associated to changes in cell physiology due to viral infection that regulates cell death, transformation, secretory pathways, cell stress response, etc [70-72]. This panorama may account for a "symbiotic evolution" of cell and virus, although viruses have much shorter generation times than cells. Studies of genomic polymorphism of HSV-1 suggest that the evolution of this virus would be very slow and host-dependent [73].

#### **5. Effect of carrageenans on the virus**

542 The Complex World of Polysaccharides

**Figure 2.** 1C3 CGN isolated from the red algae *Gigartina skottbergii* has a chemical structure similar to cellular HS. Attenuated HSV can be isolated from viral populations grown in the presence of increasing concentrations of CGN. This procedure may reflect an accelerated evolution process for HSV where

It is tempting to postulate that the driving forces of evolutionary novelty are not randomly derived from chance mutations of the genetic text, but from a precise genome editing by omnipresent viruses [58]. For decades, non-coding regions of the genome have been ignored or declared as "junk"-DNA. Recently, scientists have realized that these regions incorporate decisive higher-order regulatory functions. New research has shown that these non-coding

For a long time, viruses were interpreted as causing acute infections of susceptible organisms, using the host cellular machinery to reproduce, and achieving their lytic nature only in order to infect other cells. Although this narrative remains valid, it merely represents a case of viruses that were unable to reach persistent or chronic status of infection [61]. Most viruses, however, are stable, persistent agents that are able to establish a complex relationship with the host cell and in many cases this interaction lasts for the entire lifespan

biological modification of the viral particle can be demonstrated.

repetitive sequences originated primarily from retroviral RNA [59,60].

**4. Viruses: Friend or enemy** 

In our laboratory, viral variants of HSV-1 (strain F) and HSV-2 (strain MS) were obtained by successive passages in Vero cells under selective pressure with the 1C3 CGN (is an "hybrid" /-and partially cyclized μ/v-CGN) and ι or κ-CGNs respectively in order to test the ability of the CGN to generate resistant variants during the selection process and to study the CGN-virus modulation. Different clones were plaque purified and pretested to exclude reversion to wild type (Figure 3). 1C314-1 and 1C317-2 are viral variants derived from HSV-1 strain F (1C314-1 means, "1C3" is the type of CGN that was used for the selection of the variant, "14" number of pasagge that we chose for cloning, and "1" is the number of selected clon). 22-12, 22-13, 22-9 and 22-12 are variants derived from HSV-2 strain MS. In this case, and CGNs were used for the selection of these variants, respectively.

After the viral selection and cloning, all the variants showed a syncytial phenotype on Vero cells, this phenotype was also observed on mouse lung and genitals primary cell cultures [74,75].

**Figure 3.** Variants of HSV were obtained by successive passages in Vero cells under selective pressure with different types of CGNs with increasing concentration of them.

In order to characterize the obtained viral variants, the susceptibility to CGNs was assessed. All the viral variants showed low or middle levels of resistance to CGNs, heparin, Aciclovir (ACV), and Foscarnet (PFA) (Figure 4).

The results are shown as relative resistance (RR): IC50 viral variant/IC50 parental strain.(IC50 : inhibitory concentration 50%, is the concentration in g/ml required to reduce plaque number by 50%).

Although some variants showed 15 or 17 fold of RR, those values are not significant compared with viral variants selected with ACV (ACVp6-F and ACVp6-MS), that showed higher values, > 50 RR, after only 6 passages with ACV. (unpublished data).

**Figure 4.** Susceptibility of HSV viral variants to several compounds with known antiherpetic activity. IC50values were determinate on Vero cells by a reduction plaque assay; the RR values were calculated as (IC50: viral variants/ IC50 parental strain). Kappa: CGN; iota: CGN; ACV: Aciclovir; PFA: Foscarnet.

Mucosal surface represents the primary site for replication and spread of several viruses including HSV type 1 or 2. In order to resemble natural routes of infections, virulence of the viral variants was assessed in a mouse model by two routes of infection, intravaginal or intranasal using either BALB/c or C57BL/6 mice.

544 The Complex World of Polysaccharides

[74,75].

After the viral selection and cloning, all the variants showed a syncytial phenotype on Vero cells, this phenotype was also observed on mouse lung and genitals primary cell cultures

**Figure 3.** Variants of HSV were obtained by successive passages in Vero cells under selective pressure

In order to characterize the obtained viral variants, the susceptibility to CGNs was assessed. All the viral variants showed low or middle levels of resistance to CGNs, heparin, Aciclovir

The results are shown as relative resistance (RR): IC50 viral variant/IC50 parental strain.(IC50 : inhibitory concentration 50%, is the concentration in g/ml required to reduce

Although some variants showed 15 or 17 fold of RR, those values are not significant compared with viral variants selected with ACV (ACVp6-F and ACVp6-MS), that showed

> 1C3141 1C3172 ACVp6F K2212 K2213 I229 I2212 ACVp6MS

Kappa Iota Heparin ACV PFA

Compound

**Figure 4.** Susceptibility of HSV viral variants to several compounds with known antiherpetic activity. IC50values were determinate on Vero cells by a reduction plaque assay; the RR values were calculated as (IC50: viral variants/ IC50 parental strain). Kappa: CGN; iota: CGN; ACV: Aciclovir; PFA: Foscarnet.

higher values, > 50 RR, after only 6 passages with ACV. (unpublished data).

with different types of CGNs with increasing concentration of them.

(ACV), and Foscarnet (PFA) (Figure 4).

Relative Resistance

(RR)

plaque number by 50%).

The HSV-1 variants were avirulent for BALB/c mice infected by intravaginal route, although the parental strain F was highly lethal (Table 3). The attenuation of the variants correlated with low levels of pro-inflammatory cytokines (IL-6 and TNF-α) in vaginal lavages with respect to the parental strain, despite that viral titers were similar between the viral variants and the F strain. Nevertheless, the variants were highly lethal for BALB/c mice inoculated by the intranasal route, with a generalized organ spreading of virus.

On the other hand all the clones of HSV-2 were less virulent for mice intravaginally and intranasally inoculated, particularly for C57BL/6 mice, whereas MS strain produced 100% of mortality. In contrast to HSV-1 variants, the attenuation correlated with high levels of proinflammatory cytokines (TNF- and IL-6) detected in vaginal lavages (Table 3).

Despite the differences in the virulence of variants, the levels of infectivity in the site of inoculation did not differ from those observed for the parental strains. These results suggested that the observed attenuation was not due by a lower viral replication and it could be explained by a differentiated immunological response.


**Table 3.** Characterization of HSV variants obtained by selective pressure with CGN. Viral variants obtained under pressure of selection with carrageenans were characterized regarding their phenotype, *in vivo* virulence for BALB/c mice and immune response. The parental strains were also assessed for final comparison.

Moreover, by the same methodology, variants of HSV-2 obtained by selective pressure with nonsulfated compounds (from the condensation of mandelic acid) have been obtained. Viral variants were not syncytial and showed resistance to the same drug, heparin and the carrageenan 1C3 in the order of 2.6 to 6.7 times with respect to the control virus. However, in vivo, these variants showed no difference of pathogenicity and mortality with the parental strain.

#### **6. The evolutive beliefs**

About seven hundred and fifty million years ago, the first multicellular organisms appeared on earth. Since then, cells from multicellular organisms have found a way to become smarter. Multicellular life forms were initially isolated communities or "colonies" of unicellular organisms. However, the evolutionary advantages of living in a community soon led to communities composed of millions and billions of individual cells socially interactive. The evolutionary trend towards increasing complexity in the community is but a reflection of the biological imperative of survival. The better an organism perceives the surrounding environment, the more chances to survive. Unfortunately, we "forget" that cooperation is conveniently necessary for the evolution when Charles Darwin brought out a radically different theory about the origin of life. One hundred and fifty years ago, Darwin concluded that living organisms are involved in a constant "struggle for survival". In the final chapter of The Origin of Species by Means of Natural Selection, or The maintenance of favored races in the struggle for existence, Darwin speaks of an unstable "struggle for existence" and that evolution is conditioned by "the war of nature, from famine and death". This concept is related to the Darwinian notion that evolution occurs at random [76]. While Darwin is the most famous evolutionist, the first scientist to consider evolution as a scientific fact was the distinguished French biologist Jean Baptiste de Lamarck [77,78]. Even Ernst Mayr, the principal agent of the "neo-Darwinism," a modernization of Darwin's theory that incorporates molecular genetics of the twentieth century, admits that Lamarck was the pioneer [79]. Lamarck presented his theory not only fifty years before Darwin, but also offered a far less violent theory of evolutionary mechanisms. Lamarck's theory suggests that evolution is based on a cooperative and "instructive" interaction between organisms and the environment that allows living things to survive and evolve in a dynamic world. His idea was that organisms acquire and pass on the necessary adaptations to survive in a changing environment. One of the reasons why scientists are rethinking the theories of Lamarck is that evolutionists continue to remind the invaluable role of cooperation in maintaining life in the biosphere. In the book Darwin's Blind Spot written in 2002 by British physicist Frank Ryan has been recorded a number of these relationships [80]. Today, the understanding of cooperation in nature is much deeper than that obtained by simple observation. Biologists are increasingly aware that the animals have coevolved with different sets of microorganisms necessary for a healthy and "normal" development [81]. Recent advances in genetics have revealed an additional mechanism of cooperation between species. It has been discovered that genes are shared not only among individual members of a species (sexual reproduction) but also between members of different species. The distribution of information through gene transfer accelerates the process of evolution, in view that organisms can learn lessons "learned" by others [82-85]. Because of this distribution of genes, organisms can no longer be considered as isolated entities, there are no walls between species [86]. The distribution of genes is not a "by chance" mechanism. Is the method that nature uses to increase the survival of the biosphere. Genes are nothing more than a physical memory of the experiences learned by the organisms, the exchange of these genes between species spreads these "memories" and, consequently, influences the survival of all organisms that constitute a living community. Timothy Lenton has shown that evolution is more dependent on the interaction between species than on the interaction among individuals of the same species [87]. The evolution thus becomes a question of survival of fittest groups, not of individuals better adapted.
