Evolution of Viruses - Zhdanov V. M. 1990

Special Section
Flaviviruses

Flaviviruses, which were classified within the genus Togavirus in the 4th edition of the virus nomenclature Classification [Mathews R., 1982], were later separated into an independent family comprising a single genus [Westaway E. et al., 1985]. This extensive genus encompasses 63 Viruses, while two species (the mosquito Cell-fusing agent and the simian hemorrhagic fever agent) remain unclassified at the genus level.

Virions are small in size (45 nm), featuring a single envelope glycoprotein and two other Proteins: a core protein (C) and a membrane-associated protein (M). The infectious RNA of these viruses has a sedimentation coefficient of 44S and a Molecular Weight of 4 x 10⁶, with an m⁷G-cap at the 5'-end, but lacks a poly(A) tail at the 3'-end. It is infectious and Functions as mRNA. The Gene order is 5'-C—M—E (with ellipses denoting 3—4 non-structural proteins whose synthesis they encode). The 3'-termini of flavivirus RNA molecules are conserved, forming a characteristic Secondary Structure with large complementary regions and several loops [Brinton M. et al., 1986]. Analysis of the N-terminal RNA sequences of yellow fever, St. Louis encephalitis, and dengue 2 viruses revealed that their Glycoproteins are 52—60% conserved and 40% invariant. Nucleocapsid proteins are less conserved (25% Homology). These findings suggest that the entire group of viruses evolved from a common ancestor via divergence [Bell J. et al., 1985]. The yellow fever virus genome consists of 10,862 NUCLEOTIDES, features a cap structure at the 5'-end, and lacks a poly(A) sequence at the 3'-end of the RNA molecule. The non-coding regions at the 5'- and 3'-termini contain 118 and 511 nucleotides, respectively (Fig. 13). Polyprotein Cleavage is presumably mediated by a viral protease [McGeoch D., 1986].

The family Flaviviridae (Flaviviridae) includes a single genus of the same name (Flavivirus), which comprises A large number of viruses. Most of these viruses share serological relationships. Based on their vectors, they are categorized into mosquito-borne, tick-borne, and those with unknown vectors. However, this division is rather conventional, as some species can be transmitted by both mosquitoes and ticks.

Flavivirus virions resemble those of Togaviruses and consist of a nucleocapsid (30 nm in diameter) with cubic Symmetry and an outer envelope bearing peplomers. Virion diameter is 40–50 nm. The Genome is a single-stranded positive-sense RNA with a molecular weight of 3.8x10⁶–4.2x10⁶. Unlike togaviruses, the poly(A) sequence at the 3'-end of the RNA molecule is short. Little is known about the Introduction/11.html">Secondary structure of the genome. Flaviviruses contain three structural proteins: the capsid protein C or V2 (13,500) and two glycoproteins, E or V3 (53,000) and M or VI (8,700). Antigenic properties and The production of protective Antibodies are associated with the M glycoprotein located on the virion surface.

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Fig. 13. Genome STRUCTURE OF THE yellow fever virus. The localization of protein-coding regions is shown. Regions encoding unidentified non-structural viral proteins are designated as ns.

The Initial Stages of Replication presumably involve the contact of virions with cell Membrane Receptors, viropexis, and the fusion of viral and cellular membranes, followed by the uncoating of virions, disruption of the nucleocapsid, and release of viral RNA into the Cytoplasm. Subsequent stages of replication are less clear; in particular, the polyproteins characteristic of the non-structural and structural regions of togaviruses have not been found in flaviviruses. It is possible that individual genes or gene groups lack distinct start codons and stop signals, which are typical for both non-structural and structural protein genes.

The genomic strategy of flaviviruses differs from that of togaviruses. Virion RNA is translated in its entirety, without The formation of subgenomic RNA for the synthesis of structural proteins, with each gene being translated separately. Whereas in togaviruses structural genes are localized near the 3'-region of the genome, in flaviviruses they are situated at the 5'-end. Differences also exist in virion assembly: virions egress into cisternae rather than through Plasma Membranes. Virion morphogenesis takes place on internal cellular membranes, where budding also occurs within The Endoplasmic reticulum.

The molecular weights of the structural proteins are 51,000–59,000 (E), 13,000–16,000 (C), and 7,000–9,000 (M).

Glycoprotein E functions as a hemagglutinin. Non-structural proteins have molecular weights of 10,000 (NV1), 17,000–19,000 (NV2), 19,000–21,000 (NV21/a), 24,000–32,000 (NVX), 44,000–48,000 (NV3), 65,000–75,000 (NV4), and 91,000–98,000 (NV5). All of them differ from one another and from structural proteins. Among the listed proteins is a polymerase complex.

All flaviviruses are typical arboviruses, using warm-blooded vertebrates (mammals, birds) and Arthropods—which also act as vectors and transmit the virus transovarially—as hosts. Viruses are generally divided into three groups: mosquito-borne, tick-borne, and those with unknown vectors. This division is, of course, pragmatic, given the complex antigenic cross-reactivity within the genus.

To understand the potential evolutionary pathways of flaviviruses, mention should be made of two other groups (families) of positive-sense enveloped RNA viruses: togaviruses and Coronaviruses. All of them infect higher animals and have no counterparts among plant, fungal, or prokaryotic viruses. However, as demonstrated by the analysis of togaviruses and Picornaviruses, evolutionary links can be highly unexpected. Given the similarities between flaviviruses and togaviruses, evolutionary connections between them may be hypothesized, but a definitive resolution of this question requires more extensive studies of the genome and non-structural proteins.

Certain evolutionary pathways of these viruses become clearer in light of their ecology, and much of what applies to togaviruses is also valid for flaviviruses [Zhdanov V. M., Lvov D. K., 1984]. Currently, dengue fever is the most widespread flavivirus infection globally. For nearly two centuries, this infection was viewed as a mild ailment resulting from acclimatization to a hot climate. Its viral Etiology was established in the early 20th century. Dengue fever was considered a typical anthroponotic infection with viral Circulation between humans and mosquitoes, predominantly Aedes aegypti. However, data indicate the existence of natural foci of infection involving viral circulation between Aedes albopictus mosquitoes and monkeys. In Southeast Asia, virus-neutralizing antibodies have been detected in the lizard Calotes versicolor of the family Agamidae, and several virus strains have been isolated from monkeys in virtually uninhabited mountainous regions.

Since the 1940s, the geographic range of the pathogen has expanded dramatically. It currently encompasses equatorial, subequatorial, and tropical regions in the Americas, Africa, and Asia, home to over 1 billion people. Since the 1950s, the disease has frequently manifested (especially among children) with hemorrhagic syndrome and Shock. The fatality rate can reach 30% [Hotta S., 1978]. In Southeast Asian countries, dengue fever has become a major cause of pediatric hospitalization and, occasionally, a leading cause of mortality [Halstead S., 1980]. Since 1977, the epidemiological situation has also sharply worsened in the Caribbean basin.

The rise in dengue incidence typically follows an increase in vector populations, primarily A. aegypti, which in turn correlates heavily with ecological shifts caused by wars, refugee movements, urban growth, and declining urban living conditions. For instance, during the U.S. intervention in Vietnam, over 2 million people contracted dengue between July and September 1960 alone. An epidemic typically begins with isolated cases in major cities, followed by an explosive surge in morbidity and the spread of the infection to smaller towns and rural areas [Halstead S., 1980].

Hemorrhagic syndrome and shock usually develop in individuals who, prior to infection, possessed active Immunity (resulting from a previous infection with a different virus serotype) or passive immunity (maternal antibodies). The immunological origin of these syndromes is virtually undisputed. They occur most frequently when individuals previously infected with virus serotype 1, 3, or 4 are infected with serotype 2. At present, vector control remains the sole method for combating dengue fever. The complexity and high cost of such measures explain why the epidemic process in endemic Regions of the world remains uncontrolled at this stage.

It should be noted that until recently, various serotypes of the dengue virus had distinct geographic distributions. However, a more precise geographic zoning of the disease was achieved through restriction fragment length polymorphism analysis and monoclonal antibody techniques. As a result of these studies, the dengue virus was subdivided into 5 groups that correlated with their geographic distribution (New Guinea, Burma, Puerto Rico, Jamaica, the Philippines).

Some researchers [Dubinin V. B., 1958] suggest—though other specialists disagree—that two- and three-host patterns of tick parasitism evolved secondarily as an adaptation to unfavorable seasons and harsher conditions at higher latitudes. One-host ticks cannot serve as efficient hosts and vectors for viruses. As one moves from the equator to the poles, climatic factors (primarily a decrease in the sum of effective temperatures) lead to a sharp decline in mosquito-adapted togaviruses and, conversely, an increase in viruses ecologically associated with ixodid ticks, primarily flaviviruses. In the Pliocene, the range of Ixodes kashmiricus was significantly broader than today, likely occupying the mid-altitude forest zones of Central Asia [Filippova N. A., 1973].

A representative of the northern branch of the subgenus Ixodes pavlovskyi is a relict of the Pliocene fauna. Its range, starting from the Late Pliocene and particularly during the Pleistocene, contracted sharply under pressure from taiga expanding into the more thermophilic and humid broad-leaved Pliocene forests, and currently corresponds only to the southern portion of the range of I. persulcatus. In the post-glacial period, the range likely expanded again. The ESTABLISHMENT OF THE I. persulcatus range is linked to The Development of the taiga landscape. It is the most widespread and abundant species of the group. In the Late Pliocene, the species' range was probably restricted to eastern regions, expanding dramatically westward only during the glacial period [Filippova N. A., 1971].

Thus, among tick-borne vectors, only the primary tick-borne encephalitis vector I. persulcatus traces its origin to the taiga landscape. Furthermore, the formation of its range is relatively recent compared to other members of this phylogenetic group within the subgenus Ixodes. When its range was forming, foci of mosquito- and bird-associated togaviruses likely already existed in southern regions, such as Africa and Southeast Asia. During spring bird Migrations, viruses were systematically introduced via East Asian and Indo-Asian migratory flyways in the east, and East European flyways in the west, into the ranges of I. ricinus and I. persulcatus; some of these viruses could adapt to these tick species. Subsequent evolution may have thus given rise to a genetically stable tick-borne encephalitis virus population. Given the differences in the evolution of I. ricinus and I. persulcatus, it is also easier to envision differences between the eastern and western portions of the viral population, which are of a species-specific nature [Votyakov V. I. et al., 1978].

Experiments have demonstrated the replication of mosquito-associated alpha- and flaviviruses within tick bodies [Nosek J., 1980]. Ixodid ticks, particularly hard ticks, play an increasingly important role in the reservation of togaviruses when moving from tropical and subtropical zones to temperate and subarctic climatic belts. This is facilitated by a complex of biological and ecological traits: prolonged developmental stages, feeding of different life stages on various hosts, transstadial and transovarian transmission of togaviruses, and overwintering in diapause. All of this helps tick-adapted viruses overcome the Temperature barrier and spread to the Limits of the respective tick species' ranges. Data on the reservation of West Nile flavivirus [Lvov D. K., Ilyichev V. D., 1979] and Sindbis alphavirus [Kostyukov M. A. et al., 1981]—introduced into the USSR by migratory birds—by ixodid ticks confirm this course of events.

On the continental territory of the USSR north of the I. persulcatus range boundary, ixodid ticks are absent. Here, the warm season with temperatures effective for viral replication is extremely short. Consequently, mosquitoes, despite their immense Abundance, generally cannot sustain viral circulation. However, the coastal and insular regions of the northern temperate and subarctic zones host massive nesting colonies of marine seabirds parasitized by large numbers of Ixodes putus ticks. A prolonged metamorphosis cycle (up to 6–8 years) can provide the sum of effective temperatures required to complete the extrinsic incubation period of viruses in ticks. Survival during tick diapause ensures viral overwintering. Except during their active period (1–2 months), ticks reside in leaf litter and rock crevices at a depth of 10–40 cm, where temperatures rarely drop below 5 °C. Tick density across all developmental stages here reaches 20,000 per 1 kg of litter. Under specific conditions, this can drive extremely active viral circulation should certain viruses adapt to I. putus. These concepts formed the basis for predicting the existence of viral foci in harsh climatic environments. Testing this prediction led to the isolation of hundreds of strains of various viruses, including the Tyuleniy flavivirus [Lvov D. K., Ilyichev V. D., 1980]. It turned out that the virus is capable of biological transmission by mosquitoes both experimentally and under natural conditions. This enables a seasonal 'spillover' of the viral population from seabird colonies onto the mainland during the summer, involving a wide range of vertebrates as well as humans in the circulation cycle.

Finally, one can postulate the further evolution of flaviviruses, whereby in the absence of efficient vectors (mosquitoes, ticks), viruses may adapt to contact transmission among vertebrate hosts. If such evolution proceeds far enough, viruses may lose The ability to replicate in arthropods—that is, they cease to be arboviruses. An example of this is the Modoc flavivirus, which circulates among rodents in the Rocky Mountains of the USA, where harsh climatic conditions preclude the presence of mosquitoes and ticks. Experimentally, the virus cannot replicate in arthropods, not even in their cell cultures. In rodents, however, the virus causes a persistent infection that apparently lasts for the animal's lifetime. The virus is shed into the environment via milk, urine, and feces, with transmission presumably occurring via alimentary and respiratory routes. Persistent togoviral infection in vertebrates is a fairly common phenomenon [Lvov D. K., 1970]. States of anabiosis, such as mammalian hibernation, promote the development of viral persistence. Bats play a crucial role in the ecology of many togaviruses; 4 alpha- and 12 flaviviruses have been isolated from them in nature. Experimentally, bats develop prolonged viremia. During hibernation, viruses survive in the bats' bodies for months, and become reactivated as temperatures rise [Sulkin S., Allen R., 1974]. Bats play a significant role in the reservation of togaviruses, a process that may also be aided by the transplacental transmission of viruses regularly observed in bats under experimental conditions. It is possible that this very mechanism drove the evolutionary divergence of several bat-adapted flaviviruses that retained antigenic ties to other genus members while losing the capacity for arthropod replication.

It is important to note that serial passages of togaviruses in vertebrates or their cell cultures eventually lead to a reduction or loss of the virus's ability to infect arthropods via the oral (though not the thoracic) route of administration [Rosen L., 1980]. This likely explains the existence of many vertebrate-associated flaviviruses that have lost the capacity for biological transmission by arthropods. Such an evolutionary pathway can also be hypothesized for rubiviruses and pestiviruses. The rubella virus, which is closely related to alphaviruses, entered the human population during the Cytology/cytology/16.html">Early stages of human societal development and adapted to respiratory transmission. Through further evolution, the virus lost the ability to replicate in arthropods as well as in other mammals, with humans being the sole exception. Similar adaptation to domestic animals presumably occurred in pestiviruses—which currently spread via contact among pigs and cattle—and in unclassified togaviruses of mice, horses, and monkeys. A persistent type of infection in susceptible warm-blooded animals is characteristic of virtually all pestiviruses.

Thus, some flavivirus infections tend to become (or have already become) anthroponoses. Yellow fever and dengue fever have evolved into established anthroponoses, while retaining links to natural foci—as seen, for instance, in jungle yellow fever. Furthermore, many other diseases in this group (such as West Nile fever and Japanese encephalitis) can temporarily become anthroponotic infections under specific circumstances before returning to their natural foci.