Evolution of Viruses - Zhdanov, V. M. 1990

Special Section
Togaviruses

The family Togaviridae represents a group of enveloped Viruses with a positive-sense single-stranded RNA genome and is divided into three genera: Alphavirus (agents of vector-borne fevers and encephalitis), Rubivirus (rubella virus), and Pestivirus (agents of mucosal disease in animals). Several viruses belonging to this family remain unclassified, notably Lactate dehydrogenase-elevating virus of mice and carrot mottle virus.

Virions are 40–70 nm in diameter and consist of an isometric (cubic Symmetry) core surrounded by an outer lipid envelope bearing peplomers. Serological relationships exist among the viruses comprising each genus.

The togavirus genome (represented by Sindbis virus) is a single-stranded positive-sense RNA with a sedimentation coefficient of 49S and a Molecular Weight of approximately 4x10⁶. The RNA molecule consists of 11,200 NUCLEOTIDES. The 5'-end features a 7-methylguanosine cap Structure, while the 3'-end terminates in a poly(A) tail. The 5'-terminal region of the coding sequence is preceded by a regulatory sequence, followed by the non-structural protein Gene region (approximately 7,500 nucleotides) ending in termination codons and a conserved nucleotide sequence; this is followed by the structural protein coding region extending to the 3'-end (Fig. 14).

Virions contain 3 to 4 structural Proteins: the core protein C and the envelope Glycoproteins E1 and E2 (sometimes also E3). These protein molecules consist of 264, 439, 422, and 65 amino acid residues, respectively, with molecular weights of 26,000, 42,000, and 6,500. The molecular weights of viruses vary significantly across different genera, but show much less variation among different Representatives of the same genus. During Replication, 4 non-structural proteins are formed: NS70 (540 amino acid residues), NS86 (807 residues), NS60 (549 residues), and NS72 (610 residues).

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Fig. 14. Organization and expression of the alphavirus (Sindbis) genome. c — cap; An — poly(A); NT — nucleotides; ak — number of Amino Acids; p — proteins; gp — glycoproteins; NS — non-structural proteins; C — capsid protein; pb — hydrophobic signal sequence for E1; ↓ — initiation of Protein Synthesis; ↑ — termination of protein synthesis; 1 — conserved nucleotide sequences; 2 — protein Processing.

Following adsorption to cellular receptors on Cell/30.html">The Plasma Membrane (the host range of susceptible Cells is broad), viropexis occurs alongside the fusion of viral and host cell membranes. This process releases first the nucleoid and subsequently the nucleic acid, which interacts with the cellular ribosomal systems (Fig. 15). Translation then takes place in the non-structural gene region, which occupies about ²/₃ of The Genome, yielding 4 proteins whose genes are arranged sequentially on the virion RNA (NS70 — NS86 — NS60 — NS72) (Fig. 16). These proteins become part of the replication complex, and some drive the METABOLISM/31.html">Transcription of the structural portion of the genome from the 3'-end. This results in The production of subgenomic RNA (first the minus strand, followed by the coding plus strand) carrying the structural protein genes, which are arranged from the 3'-end in the following order: E1—E2 — (E3) — C.

Fig. 15. Schematic representation of togavirus entry into The Cell via receptor-mediated endocytosis.

1 — binding of Viral Particles to receptors in coated pits; 2 — formation of coated vacuoles; 3 — localization of viral particles in uncoated vacuoles and fusion of the viral envelope with the vacuolar membrane at acidic pH; 4 — release of the nucleocapsid into the Cytoplasm; 5 — uncoating of the viral genome.

Replication proceeds via the synthesis of a full-length genome-length minus strand (49S), from which the plus strand is transcribed, along with a putative subgenomic 26S RNA carrying the structural

genes. This strand (approximately 3,700 nucleotides) is also translated into a polyprotein (~130,000), which is subsequently cleaved in two stages (C, E1 + E2; E1, E2) to yield structural proteins, with E1 and E2 undergoing glycosylation. Proteolytic Cleavage likely involves a viral protease, with the capsid protein exhibiting enzymatic activity. During polyprotein processing, an oligopeptide (p6) of unclear function is produced. Nucleocapsids and structural proteins are synthesized at different sites within the cell and transported to the plasma membrane, where mature virions are assembled and released by budding.

Most togaviruses infect warm-blooded animals and are transmitted by Arthropods, within the bodies of which they also replicate [Gaydamovich S. Ya., Loginova N. V., 1982]. Only a few species are transmitted without Blood-sucking arthropods. One plant virus, carrot mottle virus, shares strong similarities with togaviruses and has been assigned to this group, which constitutes the family Togaviridae.

It is worth noting several additional details that may prove useful in understanding the evolutionary pathways of this virus family. In the latest edition of the Virus Classification [Matthews R., 1982], the family Togaviridae is divided into 4 genera: alphaviruses, Flaviviruses, rubiviruses, and pestiviruses.

Fig. 16. Schematic diagram of translation and processing of Sindbis virus non-structural proteins. The translatable region of the viral genomic RNA is shown as an Open Reading Frame, the untranslated region as a line, and nt represents the number of nucleotides. Final protein products are indicated by bold lines. Proteins nsP1–P5, generated during processing of p(1–4), are shown with dashed lines as they remain unidentified.

Alphavirus nucleic acid has a molecular weight of 4.0x10⁶–4.6x10⁶. The virions contain three structural proteins: one internal non-glycosylated protein and two outer envelope glycoproteins with molecular weights of 30,000–34,000 and approximately 50,000, respectively. The virion diameter is about 70 nm. In flaviviruses, the RNA molecular weight is similar, but the proteins differ: the nucleocapsid protein (C) is 13,000–16,000, the outer envelope glycoprotein (E) is 51,000–52,000, and a small third protein (M) is 7,000–9,000. Virion diameter is approximately 40 nm.

Rubivirus (the CAUSATIVE AGENT OF rubella) has been classified into a separate genus based on its similarity to other togaviruses coupled with its distinct ecology (transmission occurring without the intermediacy of blood-sucking arthropods). The gene order of the rubella virus [Oker-Blom C., 1984] is as follows: NH₂ = C—E2—E1 = COOH. Pestiviruses were likewise separated from the general togavirus family primarily on The basis of ecological data. In addition, several unclassified viruses are assigned to the family Togaviridae. These include, notably, lactate dehydrogenase-elevating virus, mosquito cell-fusing agent virus, and the previously mentioned plant virus affecting carrots.

In a newer classification scheme, flaviviruses have been elevated to a separate family due to differences in their genome strategy, morphogenesis, and protein profiles [Westaway E. et al., 1985]. The older classification [Porterfield J. et al., 1978], which formed the basis of the 4th general edition [Matthews R., 1982], has been superseded by a new one [Westaway E. et al., 1985]. In this classification, the family Togaviridae, as already noted, comprises 4 genera: Alphavirus (26 species), Rubivirus (1 species), Pestivirus (3 species), and Arterivirus (1 species). The characterization of the type species has been slightly revised. Different species possess varying numbers of structural proteins; for instance, Sindbis virus has 3, whereas Semliki Forest virus has 4. The molecular weights of these proteins are as follows: E1 and E2 range around 50,000 and 30,000–34,000, respectively, while E3 is approximately 10,000. Protein C forms the capsid, whereas proteins E1–E3 reside in the outer envelope, with hemagglutinating activity associated with protein E1. The core diameter is 35 nm and the virion diameter is 50 nm. Non-structural proteins—numbering 3 to 4—are generated from precursors and, in Sindbis virus, have molecular weights of 60,000, 72,000, 76,000, and 89,000. They exhibit polymerase activity, whereas autocatalytic protease activity is apparently associated with one of three non-structural precursors (molecular weights of 150,000, 230,000, and 270,000). During RNA replication, the entire strand is transcribed, whereas the synthesis of structural proteins is driven by a subvirion 26S RNA, on which the structural protein genes are arranged in the order: 5'-C—E3—E2—E1-3'.

In Sindbis virus, the 49S RNA with a sedimentation coefficient and a molecular weight of 4x10⁶ consists of 11,703 nucleotides, featuring a 5'-terminal cap structure and a 3'-terminal poly(A) tail. The first 59 nucleotides from the 5'-end are regulatory. The open reading frame for non-structural protein genes spans 7,539 nucleotides, followed by 48 untranslated bases separating it from the structural protein gene region, which comprises 3,735 nucleotides. This region terminates in a 3'-untranslated region of 322 nucleotides. Non-structural proteins are synthesized from two precursors. The first molecule consists of 1,896 amino acid residues and is cleaved into nsP1, nsP2, and nsP3; the second protein, initiating from the same translation start site, consists of 2,513 amino acid residues and harbors the nsP4 protein at its carboxy-terminus. Structural proteins are synthesized from a single precursor [Strauss E. et al., 1984].

The molecular weight of rubella virus (Rubivirus) RNA is approximately 3.2x10⁶–3.8x10⁶, with the RNA sedimenting at 40S. Its three structural proteins have molecular weights of 58,000–59,000 (E1), 42,000–48,000 (E2), and 33,000–34,000 (C); the first two are glycosylated and embedded in the lipid envelope, while the third is the capsid protein. Hemagglutinating activity is linked to protein E1. Virions are 60 nm in size, with a core of 30–35 nm. The heterogeneity of protein E2 is attributable to variable degrees of glycosylation. The molecular weights of the 4 non-structural proteins and their precursors are 200,000, 150,000, 87,000, and 75,000 [Bowden D., Westaway E., 1984].

Pestivirus species possess an RNA with a sedimentation coefficient of 38–40S and a molecular weight of approximately 4x10⁶. The molecular weights of their 3 structural proteins are 55,000–57,000 (E1), 44,000–46,000 (E2), and 34,000–36,000 (C). The virion diameter is 50–60 nm, and the core diameter is 27–35 nm.

Equine arteritis virus has been classified into a distinct genus, Arterivirus. Its RNA sediments at 48S, has a molecular weight of 4.1x10⁶–4.3x10⁶, and features a poly(A) tail at the 3'-end. Three Peptides (the first of which is definitively a glycoprotein) have molecular weights of 21,000 (E1), 14,000 (E2), and 12,000 (C). The virion diameter is 60 nm, and the core diameter is 35 nm.

Thus, upon closer scrutiny, the family Togaviridae has proven to be an even more compact group than it was when it encompassed the genus of flaviviruses.

A separate case is the lactate dehydrogenase-elevating virus of mice [Rowson K., Mahy B., 1985]. Based on its Morphology and genome strategy, this virus has been assigned to the togaviruses, forming an unclassified group alongside equine arteritis virus and simian hemorrhagic fever virus. The virus establishes a life-long persistent infection in mice, accumulating in high titers in the blood (10⁹ ID₅₀/ml), and is transmitted by blood-sucking insects. Viremia is asymptomatic, although strains causing polyencephalitis have been described. Susceptibility is restricted to two mouse species (Mus musculus, M. caroli). Other rodents are refractory, and susceptible host cells are restricted to macrophages, which are destroyed by the virus. The ORIGIN AND EVOLUTION of this virus remain a puzzle. Is it an evolutionary offshoot of togaviruses or, conversely, their precursor? Future comparative studies of this virus and other togaviruses will provide the Answers.

Let us recall that, In addition to the group under consideration, animal viruses with a polar-positive genome and a supercapsid include two other groups: flaviviruses and Coronaviruses. Aside from the aforementioned features, they share a genome structure that dictates its replication strategy: a cap structure at the 5' end and a poly(A) sequence at the 3' end of the RNA molecule. Among enveloped plant viruses, a similar structure is found in tomato spotted wilt virus, whose genome consists of 4 fragments with a total molecular weight of 7.5X106, and its virions are spherical in shape. Unfortunately, this virus has not been sufficiently studied, precluding a comparative analysis with the group of viruses under Discussion. Among non-enveloped animal viruses with a positive genome, Picornaviruses and Caliciviruses lack cap structures at the 5' ends of their RNA molecules. They have other analogs among viruses infecting Fungi, plants, and animals.

We encounter unexpected findings when turning to certain other groups of plant-infecting virions. First, The Study of defective interfering particles of Sindbis virus revealed RNAs containing deletions (and thus non-infectious) but bearing at their 5' ends an aspartic tRNA covalently linked to the virion RNA. As is well known, such structures are highly characteristic of the RNAs of many plant-infecting viruses (such as those of the yellow fever, turnip, southern bean mosaic, and tobacco mosaic virus groups). Second, a comparative study of Sindbis, brome mosaic, and alfalfa mosaic viruses demonstrated Homology among certain non-structural proteins functioning as polymerases.

It should be noted that the tobacco mosaic virus and similar viruses (15 species) of this group possess a single-stranded RNA genome with a molecular weight of approximately 2x10⁶, a cap structure m7G5'ppp⁶5'Gp at the 5' end, a tRNA-like structure at the 3' end, and rod-shaped virions with helical symmetry [Fraenkel-Conrat Н., 1981]. Brome mosaic virus is a tripartite virus with RNA segment molecular weights of 1.1x10⁶, 1.0x10⁶, and 0.7x10⁶ (totaling 2.8x106); each segment features a cap structure at the 5' end and a tRNA-like structure at the 3' end. The particles are polyhedral in shape with a diameter of 26 nm. Alfalfa mosaic virus is also tripartite, with RNA segment molecular weights of 1.1x10⁶, 0.8x10⁶, and 0.7x10⁶ (totaling 2.6x10⁶); a cap structure is located at the 5' end of the segments, and the rod-shaped particles measure 58x18 nm (B), 48x18 nm (M), and 36x18 nm (Tv); in addition, ellipsoidal particles measuring 28x18 nm (Ta) contain the Messenger RNA for the capsid protein.

Fig. 17. Comparison of the genomic RNA structures of Sindbis virus (4) and three plant viruses (1–3). Subgenomic RNAs are shown below the genomic RNA; protein-coding regions are outlined with a frame; I — initiation codon; II — terminal codon; homologous sequences are indicated by identical shading.

All three of these plant viruses, despite being heteropartite and exhibiting Different types of virion or particle symmetry, share a similar genome structure and strategy. Furthermore, within the non-structural protein region, they show pronounced homology across 3 domains. One of these, protein 2a—common to brome mosaic and alfalfa mosaic viruses—shares significant homology with the p183 protein region of tobacco mosaic virus, as well as with the nsp4 protein of Sindbis virus [H-aseloff J. et al., 1984]. The other two domains are represented by protein 1a of the mosaic viruses and the nsp1 and nsp2 proteins of Sindbis virus [Ahlquist Р. et al., 1985]. These findings also lead to the Conclusion that these domains encode the synthesis of polymerase complex proteins outside of the 4 studied viruses, which proved to be evolutionarily conservative, whereas the domains encoding structural proteins diverged to such an extent that it led to changes in their packing symmetry and even in heteropartite organization. Fig. 17 shows a diagram from the cited works illustrating these data.

Other data point to unusual crossovers in the evolution of togaviruses. Barmah Forest virus is a typical alphavirus: its morphology is characteristic of this group, the N-terminal Regions of the E1 and E2 glycoproteins exhibit significant homology (50%) with those of other alphaviruses along with serological relatedness, yet it also shows serological affinity with Umbre virus, which belongs to Bunyaviruses [Dalgrano L. et al., 1984].

The presented data strongly suggest a common ancestry for all 4 viruses. Over a long period of divergent evolution, numerous events took place: the structural Proteins of the viruses completely changed, their packing symmetry within the capsid diverged (cubic symmetry in Sindbis and brome mosaic viruses, helical in tobacco mosaic and alfalfa mosaic viruses), and heteropartiticity and correspondingly fragmented genomes developed (monopartite Sindbis and tobacco mosaic viruses, tripartite brome mosaic and alfalfa mosaic viruses, with the latter also possessing a fourth particle for mRNA "packaging"). While the plant viruses were preserved as ribonucleoprotein structures, animal alphaviruses acquired outer envelopes. Despite all these changes, the polymerase complex has remained remarkably conservative across such diverse viruses.

Naturally, the common ancestor of these viruses has not survived, and it is difficult to determine whether it should be sought among plants or animals. Perhaps, to understand the time of their emergence, it is worth noting that all the compared viruses infect only higher plants (angiosperms), and some of them (except for the tobacco mosaic virus group) are transmitted by insects, although this transmission is primarily mechanical, with the exception of the poorly understood tomato spotted wilt virus group (which persists in the larvae of insect vectors). A comparison of 13 viruses of the tobacco mosaic virus group revealed significant divergence of virion proteins, leading to the conclusion that the time of emergence of their common ancestor is comparable to the appearance of flowering plants [Gibbs A., 1980; Gibbs A. et al., 1982].

Assuming that plants are the primary "hosts" of all 4–5 groups of viruses under consideration, the most probable scenario is that they transitioned to parasitizing animals via blood-sucking arthropods. This likely occurred after these viruses had already occupied a broad ecological niche by infecting many species of flowering plants. Incidentally, the Evolution of the viral Lineage that gave rise to the togavirus family may have begun at the stage of parasitism in plants, as evidenced by the existence of a typical plant-infecting togavirus (carrot mottle virus).

The formation of togaviruses transmitted by blood-sucking arthropods evidently occurred through a complex process: by multiplying in arthropods, the viruses conquered new ecological niches in the form of new warm-blooded "hosts"—the blood meal sources for the arthropods—while these warm-blooded hosts could transmit the virus to new arthropod vectors. In this manner, viruses could pass from ticks to insects and from insects to ticks. This mechanism can account for the Abundance of alphaviruses and their global distribution. Pestiviruses arose much later, and their appearance can be linked to the domestication of animals. Under herd-housing conditions, the opportunity arose for contact transmission of the virus without the involvement of blood-sucking arthropods. Consequently, this may have enabled The Emergence of purely human infections with an airborne transmission mechanism, such as rubella.

Such are the general outlines of the possible evolutionary Pathways of the virus groups causing these infections. Let us now examine the later stages of their evolution, focusing on togaviruses, or more specifically, alphaviruses [Zhdanov V.M., Lvov D.K., 1985].

Despite substantial differences in replication, morphology, morphogenesis, and ecology, one can hypothesize a common evolutionary origin for all togaviruses. Currently, most alpha- and flaviviruses belong to the ecological group of arboviruses. The majority of these viruses are presently transmitted by arthropods via biological transmission to vertebrate animals and possess The unique ability to replicate both at the body Temperature of warm-blooded animals and at relatively low ambient temperatures during replication in the bodies of blood-sucking arthropods. Following the infection of the vector, a certain period of time must elapse during which the virus, having entered the digestive tract with blood and crossed the peritrophic membrane, multiplies in the midgut epithelium of the vector. Only after this, having penetrated the gut wall, does the virus multiply in body Tissues and accumulate in the salivary apparatus in quantities sufficient for effective infection of a vertebrate upon a bite. The extrinsic incubation period is shorter when the ambient temperature is higher. Conversely, when the temperature drops below a threshold level (approximately 18±5 °C for various viruses), viral reproduction ceases.

Consequently, the most favorable conditions for the existence of togaviruses, other things being equal, occur at a constantly high ambient temperature (around 28–30 °C). Such temperature conditions characterize the equatorial and subequatorial belts. It is natural to assume that togaviruses emerged precisely under these optimal conditions. In this regard, let us examine data on their current geographical distribution. The number of known togaviruses sharply decreases with distance from the equator and subequator toward the tropics, subtropics, and temperate zone, and this reduction occurs specifically at the expense of mosquito-borne viruses.

Thus, an Analysis of the primary ecological features and current Geographical Distribution of togaviruses points to their initial emergence in the equatorial and subequatorial climatic Zones of the Earth.

Given their mechanism of infection, togaviruses can be regarded as blood parasites. It is generally accepted that this form of parasitism is secondary and evolved from intestinal parasitism. Evidently, the majority of vertebrate blood parasites were originally parasites of the invertebrate intestine. Later, as certain arthropods shifted to blood-sucking, some of these parasites acquired The ability to penetrate the gut wall, replicate in their Organs and tissues, and be transmitted upon biting vertebrates. The viruses replicated in the cells of various tissues at the body temperature of warm-blooded animals. In other words, togaviruses adapted to a new habitat—the tissues of warm-blooded animals.

THE ORIGIN OF togaviruses from symbiont (or parasite) viruses inhabiting the intestinal walls of arthropods is supported by the following facts: 1) the presence of symbiont viruses in arthropods, particularly mosquitoes; 2) the absence of significant harm to arthropods infected with togaviruses; 3) persistent viral infection in tissues throughout the adult insect's lifespan; 4) transstadial and transovarial transmission of the virus during metamorphosis; 5) the cyclical development of arthropod viruses with an obligatory and prolonged phase of replication in the gut wall; 6) an ability to replicate at relatively low temperatures, which is unique for warm-blooded animal viruses but common for arthropod viruses. All this points to an ancient relationship and a high degree of adaptation of togaviruses to arthropods. Let us dwell in more detail on some of these arguments.

In a continuous cell line derived from Aedes albopictus mosquito larvae, Electron Microscopy revealed 5 different TYPES OF VIRAL particles, including togavirus-like ones. Using the same method, cytoplasmic crystal-like formations of viral particles, similar in size and shape to alphaviruses, were isolated from this cell line.

Following ultrasonic Treatment and density gradient fractionation in sucrose, the fraction induced plaque formation in Vero cells. Subsequent passages on BHK-21 cells revealed a gradually developing cytopathic effect (CPE). The agent is serologically similar to Chikungunya virus [Brinton M., 1980]. In Australia, a clone of the A. albopictus cell line resistant to Semliki Forest virus infection was discovered. It turned out that the clone had been infected with this virus even prior to inoculation. Finally, a cell-fusing agent (CFA) was isolated from the culture medium of an A. aegypti mosquito cell line, which is detected only upon infection of the A. albopictus cell line [Stollar V., 1980]. In the cells of this line, the agent induced syncytium formation 48–72 hours post-infection. Virus maturation occurred via budding through intracellular membranes. The results of studying the physicochemical properties, morphology of this virus, its genome, and structural proteins allow this agent to be classified as a togavirus [Stollar V., 1980]. In size and morphology, it resembles flaviviruses, but their budding patterns differ. The agent lacks hemagglutinin and shows no antigenic relationships with any flaviviruses. Therefore, the systematic position of the virus among other togaviruses has not yet been determined.

The presented data indicate the presence of symbiont togaviruses in mosquitoes. Identifying them is in some cases extraordinarily difficult. All alphaviruses and many flaviviruses can also be regarded as arthropod symbiont viruses. This conclusion can be drawn from an analysis of The Nature of the interactions between these viruses and arthropods.

Experimental studies of virus reproduction in vectors established that viruses persist in various tissues (particularly the gut) throughout the arthropod's lifespan without causing harm in the majority of cases. Thus, togaviruses typically induce an asymptomatic persistent infection in arthropods. This was demonstrated using the model of Japanese encephalitis virus infecting Culex pipiens pallens mosquitoes, as well as in studies of Sindbis virus infection in Aedes albopictus mosquitoes [Condrea L., Brown D., 1986]. It must be noted, however, that Semliki Forest virus can cause destructive Changes in the salivary gland cells of Aedes aegypti mosquitoes.

Using fluorescent Antibodies, several authors studied The Fate of Japanese encephalitis virus in the body of Culex tritaeniorhynchus summorosus mosquitoes. It was established that the virus initially multiplies in the midgut, then in the fat body, and finally in the Salivary Glands and other Organs of the mosquito. Latent infection was observed throughout the mosquito's lifespan. Similar data on the dynamics of this virus's reproduction in C. pipiens pallens were obtained using electron microscopy.

Experimental latent infection with St. Louis virus and western equine encephalomyelitis (WEE) virus in C. quinquefasciatus and A. tarsalis mosquitoes under diapause conditions was observed throughout the overwintering period (5–8 months), and WEE virus was isolated from overwintering C. tarsalis mosquitoes. This represents one of the ways togaviruses survive the winter period as well as the dry season in arid regions—namely, periods critical for the survival of viral populations.

Ticks infected with togaviruses also regularly exhibit The Development of persistent latent infection.

Togaviruses are capable of replication upon parenteral infection of non-blood-sucking arthropods as well. The reproduction of Japanese encephalitis virus has been established, for example, upon infection of beetles and moths, and that of Sindbis virus upon infection of Drosophila. Two alphaviruses are capable of replicating in the bodies of Oecaeacus vicarius bugs parasitizing swallows, which serve as specific vectors for these viruses [Chamberlain R., 1980; Rush W. et al., 1980].

When infected parenterally, non-hematophagous male mosquitoes are just as susceptible to viruses as hematophagous females. Mosquito larvae are easily infected with togaviruses via the alimentary route, with subsequent transmission of the virus to the imago during metamorphosis [Rosen L., 1980]. All of this points to a strong adaptation of togaviruses to arthropods, and varying susceptibility to infection has been demonstrated even among different strains of the same species.

A wealth of data has been accumulated regarding the patterns of togavirus reproduction in arthropod tissue cultures. The cell lines most widely used for this purpose are derived from A'edes albopictus larvae, A. aegypti embryos, as well as from various species of Aedes and Anopheles larvae - [Pudney М. et al., 1970], and C. tritaeniorhynchus. Some tick-derived cell lines have also been established, notably the RML-14 line from Dermacenter parumapertus, which Supports robust growth for many tick- and mosquito-borne alpha- and flaviviruses.

As a rule, the vast majority of cells in these cultures become infected. The latent period in this case is longer (5–6 hours instead of 3–4 hours) compared to that in warm-blooded animal cells. When togaviruses are grown in arthropod cell cultures, certain peculiarities are observed in THE SPECTRUM OF CARBOHYDRATES (absence of sialic acid, decreased galactose content) and Lipids (altered phospholipid ratios), but not in RNA and structural proteins [Stollar V., 1980]. The production of a low-molecular-weight polypeptide that inhibits virus production has also been detected. This substance exhibits both cellular and Viral Specificity, which highlights the significant specificity of togavirus replication in arthropod cell systems.

Alphaviruses, which are highly pathogenic for warm-blooded animal cells, do not induce a cytopathic effect in arthropod cell systems. Conversely, flaviviruses—much like the cell-lysing virus mentioned above—typically induce extensive cell lysis. However, this does not always occur; even when a cytopathic effect develops, islands of surviving cells remain, which eventually, within a few days or weeks, lead to the restoration of the cell culture. In such instances, as is consistently the case with alphavirus infections, a persistent infection develops over many months, lasting virtually until the end of cultivation [Stollar V., 1980]. Notably, the cytopathic effect is more pronounced at 34–37 °C and less pronounced at 28 °C (the ambient temperature in the equatorial climate zone). Conversely, virus production is higher at 28 °C compared to 34 °C. As a result of such persistent infection, the pathogenicity of the virus for mice decreases in A number of cases, occasionally correlating with a small-plaque phenotype. Cell cultures with persistent infection are resistant to superinfection solely by the homologous virus.

These findings provide a solid basis for the conclusion that arthropods serve not only as vectors, but also as permanent "hosts" for togaviruses. Long-term survival of virus populations is possible within a dual arthropod-virus parasitic system, featuring vertical transmission of the virus during metamorphosis, sexual transmission, and alimentary infection of mosquito larvae with subsequent transmission to the imago. Consequently, all togaviruses isolated from arthropods (which constitute the absolute majority) can be regarded as symbionts or, in some cases, parasites of these creatures. The periodic (and very brief, from the virus population's perspective) incorporation of a third member into this parasitic system—a vertebrate (typically warm-blooded) host—is likely beneficial to the virus population, as it enriches its gene pool. Sometimes, as will be shown below, the involvement of warm-blooded animals in the Circulation of togaviruses alters the course of their evolution and leads to the colonization of new ecological niches by the virus population [Lvov D. K., 1970; Lvov D. K., Lebedev A. D., 1974].

As noted above, climatic conditions—temperature first and foremost—in the equatorial and subequatorial belts are the most favorable for the survival of togavirus populations. Under these conditions, the vast majority of them are ecologically associated with mosquitoes [Cornet М. et al., 1980]. It is logical to assume that these very insects served as the primary "hosts" of togaviruses. Over time, some of these viruses acquired the ability to accumulate in the salivary glands and infect birds and other warm-blooded animals during blood-feeding. This evolutionary stage is associated with the acquisition by the viruses of the rct⁺42° genes (the ability to replicate at avian body temperatures), as well as V⁺ and Pp (the capacity to induce viremia in warm-blooded animals following peripheral infection). However, the ability to replicate at relatively low ambient temperatures (rct⁺ 20°) had to be preserved. From this evolutionary milestone onward, togaviruses functionally became arboviruses. In the equatorial and subequatorial climates, ixodid ticks could hardly compete with mosquitoes as primary hosts. Results obtained in recent years corroborate much earlier data regarding the ability (at least of flaviviruses) to be transmitted transovarially during mosquito metamorphosis.

The wide geographic distribution of the Sindbis virus has increased the heterogeneity of its population. Genetic studies of isolates of this virus obtained from various regions and different vectors have demonstrated [Olson K., Orent D., 1985] the existence of two viral groups: the Palaearctic-Ethiopian and the Oriental-Australian. Viruses were isolated from both mosquitoes and ticks. In light of these findings, the emergence of Karelian fever becomes understandable, as its causative agent diverged from the broader pool of the heterogeneous Sindbis virus population.

In August and September of 1981, numerous cases of febrile illness accompanied by rashes and arthralgia broke out in the Karelian ASSR. In some instances, the disease took on a chronic course, leading to the development of arthrosis and disability. The Etiology of this condition, designated as Karelian fever, was successfully deciphered [Lvov D. K. et al., 1982; Niklasson B. et al., 1984]. The causative agent (an arbovirus) belongs to the genus Alphavirus within the Sindbis virus antigenic complex. While the widespread African Sindbis virus typically causes sporadic, mild illnesses, an epidemic outbreak occurred in Karelia. Although the vector-borne transmission route of the disease is beyond doubt, the full range of vectors (as well as vertebrate hosts) remains to be identified. Alongside the Karelian ASSR (situated on the border of the northern and middle taiga landscape zones), the disease simultaneously appeared in ecologically similar regions of Finland (Pogost disease) and Sweden (Ockelbo disease). The unexpected emergence of an epidemic situation associated with a virus of clearly African origin in the subpolar areas of Scandinavia can be attributed to the avian transport of a portion of the viral population, followed by its subsequent evolution and adaptation to unusual and harsh conditions. It is worth noting that the East European and West European migration routes of birds that nest in Northern Europe and winter in Africa both pass through the Scandinavian regions endemic to Karelian fever. This example highlights the ongoing evolution—in this case, of an alphavirus—which can precipitate a sharp and unexpected escalation of the epidemiological situation.

The West Nile virus is regularly brought into our country by migratory birds from Africa, resulting in the formation of seasonal bird-mosquito-virus foci. A fraction of the viral population has adapted to biotopes on the islands along the Caspian Sea coast, maintaining a circulation cycle of the herring gull (Larus argentatus) — argasid tick (Ornithodorus capensis) — virus type. These islands host gull colonies parasitized by ticks, while mosquitoes are entirely absent.

Argasid ticks sustain persistent natural foci of West Nile fever, though they do not manifest epidemiologically. When the gulls depart from their nesting sites in the autumn, the virus spreads along the coastline, and mosquitoes become "recruited" into its circulation cycle. This drives the further dissemination of the virus and its incorporation into the circulation systems of other wild and, subsequently, domestic animals, ultimately triggering an epidemic situation.

Virus evolution has progressed significantly in the arid regions of Central Asia. Several strains of a novel Karshi virus have been isolated from argasid and ixodid ticks, exhibiting only a one-way antigenic relationship with the West Nile virus. These foci are quite isolated. The connection between this portion of the population and the main West Nile virus population was likely severed long ago, and evolution under these specific conditions has culminated in the emergence of a new species. Serological data indicate that under natural conditions, the Karshi virus is capable of causing febrile illness in humans. When ticks are experimentally infected, the virus accumulates across various tissues and organs, including the salivary glands. Furthermore, under experimental conditions, the virus retains its capacity for reproduction within the mosquito Organism, accumulation in the salivary glands, and biological transmission via blood-feeding. These findings underscore the conventional nature of dividing togaviruses into "mosquito-borne" and "tick-borne" categories. Everything depends on ecological conditions, which ultimately dictate the epizootic and epidemiological situation.