Viruses and Viroids: A Special Taxonomy

They lack cellular structure; obligate intracellular parasites

The first and most important thing to grasp: viruses are fundamentally different from all other known living beings. Unlike bacteria, fungi, plants, and animals, viruses do not have a cellular structure. They represent a noncellular form of life. In essence, a virus is a nucleoprotein, that is, a complex consisting of nucleic acid (genetic material) and protein (Agrios, 2005). The protein shell, called the capsid, protects the genetic material and ensures its delivery into the host cell.

This gives rise to a second fundamental feature: viruses are obligate intracellular parasites. This means they are absolutely incapable of reproducing outside a living cell. They have no metabolism of their own; they cannot independently synthesize proteins or produce energy. To reproduce, a virus must penetrate a host cell (whether plant, animal, or bacterium) and completely reprogram its metabolic resources to produce viral components (Agrios, 2005). A virus does not "eat" the cell or kill it with toxins, as many bacteria do. Instead, it uses its resources, occupies intracellular space, and disrupts normal cellular processes, which ultimately leads to disease (Agrios, 2005).

Contested status: living or nonliving?

This feature gives rise to one of the most interesting and long-standing debates in biology: are viruses alive? On the one hand, they possess genetic material, are capable of replication (reproduction), and evolve, which makes them akin to living organisms. On the other hand, they are not cells, have no metabolism of their own, and cannot reproduce independently, which makes them similar to complex chemical compounds.

Modern science proposes to view viruses not as "living" or "nonliving" in the usual sense, but as a special form of existence of genetic information (Koonin, 2021). They lie on the boundary between the living and nonliving worlds, and this unique position makes them an indispensable object for studying fundamental biological questions, including the origin of life (Koonin, 2021).

Huge diversity and ecological role

Despite their apparent simplicity of structure, viruses are an incredibly diverse group. According to estimates, their total number exceeds 2,000 species known to science, and new ones are discovered almost monthly (Agrios, 2005). They infect all forms of life: from bacteria and archaea to plants, fungi, and animals. Almost half of the known viruses cause diseases in plants (Agrios, 2005).

The ecological role of viruses is enormous and multifaceted. They are a most important factor regulating the population sizes of their hosts. In aquatic ecosystems, for example, viruses play a key role in controlling phytoplankton abundance, influencing global biogeochemical cycles. Viruses are also a powerful engine of evolution, since they participate in horizontal gene transfer between organisms (Koonin, 2021).

Importance for agriculture: crop losses, quarantine, certification

For us, as specialists in the agricultural sphere, the importance of viruses cannot be overestimated. Viral diseases of plants cause colossal damage to agriculture worldwide. They can lead to yield reductions of 30–50%, and in some cases to complete crop loss (Kreuze et al., 2020).

Viral diseases cause damage by reducing not only quantity but also product quality. For example, viral infections can make fruits unsuitable for sale or processing. In addition, viruses create serious problems for international trade. Many viruses are quarantine objects, and their detection in a shipment of planting material or seeds can lead to an import ban and destruction of the entire shipment.

That is precisely why accurate identification of viruses has critical practical importance. Without it, neither effective diagnosis (making the correct diagnosis), nor certification of planting material (confirmation of its health and purity), nor the development of quarantine strategies (preventing the spread of dangerous pathogens) is possible (Blancard, 2012).

In the next chapter, we will examine in detail how the scientific community attempts to organize this enormous diversity of viruses using the official taxonomic system — ICTV.

ICTV — the official system for classifying viruses

ICTV was created in 1966 as a committee under the International Union of Microbiological Societies (IUMS). Its main task is the development and maintenance of a unified, universal taxonomic scheme for all viruses, including viroids and some groups of satellite viruses (King et al., 2012). It is ICTV that is the body that makes decisions on the creation of new taxa (groups) and assigns them official names. ICTV decisions are binding for all virologists worldwide, which ensures stability and continuity in scientific literature and practical diagnostics.

Principles: a hierarchical system based on genome, replication, structure

The ICTV taxonomy, like any biological classification, is hierarchical. However, unlike the classification of plants or animals, where morphological and evolutionary traits form the basis, virus taxonomy is based on a complex of molecular-biological characteristics. The main criteria for assigning a virus to a particular taxon are:

1. Type and structure of the genome: DNA or RNA, single-stranded or double-stranded, linear or circular, segmented or not, strand polarity (plus or minus).

2. Replication strategy: The mechanism by which the virus copies its genome and synthesizes mRNA. This criterion is closely related to the Baltimore classification, which we will discuss below.

3. Virion structure: Presence or absence of a lipid envelope, type of capsid symmetry (helical, icosahedral), size and shape of particles.

4. Biological properties: Host range, transmission routes, pathogenicity.

The modern ICTV taxonomy, reflected in the ninth and subsequent reports, includes the following main ranks (King et al., 2012):

• Realm: The highest rank, introduced relatively recently. It unites viruses that share a common evolutionary origin, traced by conserved "marker" genes, for example, genes for replicase or capsid protein. As of 2021, several such realms are distinguished: Riboviria (all RNA viruses and reverse-transcribing viruses), Monodnaviria (single-stranded DNA viruses), Duplodnaviria (double-stranded DNA viruses, including herpesviruses and bacteriophages), and others (Koonin, 2021).
• Kingdom: An intermediate rank introduced for a more accurate reflection of evolutionary relationships within large realms.
• Phylum: A rank uniting related classes.
• Class: A rank uniting related orders.
• Order: A rank uniting related families. For example, the order Picornavirales unites several families of small RNA viruses.
• Family: A rank uniting related genera. Family names are always capitalized and end in -viridae. For example, Potyviridae, Tobamoviridae.
• Genus: A rank uniting closely related species. Genus names are also capitalized and end in -virus. For example, Potyvirus, Tobamovirus.
• Species: The basic unit of taxonomy. A species is a group of viruses having similar biological properties and a high level of genetic relatedness. Species names are usually not Latinized but are a descriptive phrase, often including the host name and symptom. For example, Tobacco mosaic virus.

Differences from binomial nomenclature: names are often descriptive, not Latinized

The most important difference between viral nomenclature and the classical Linnaean binomial system (genus + species) is that the names of viral taxa are not Latinized. They are either acronyms (for example, CaMV — Cauliflower mosaic virus) or descriptive English phrases. As we have already noted, family and genus names are standardized by endings, but the word roots themselves can be anything. This makes viral nomenclature more flexible, but sometimes less intuitive for a nonspecialist.

Examples: Potyvirus, Tobamovirus, Cucumovirus, Closterovirus

Let us consider several key genera of plant viruses that you will constantly encounter in your practice:

• Potyvirus: The largest genus of the family Potyviridae. The name comes from Potato virus Y. These are filamentous viruses with a (+)ssRNA genome, transmitted by aphids. It includes many economically important pathogens, such as cucumber mosaic virus and plum pox virus (Agrios, 2005).
• Tobamovirus: Genus of the family Virgaviridae. Name from Tobacco mosaic virus. These are rod-shaped viruses with a (+)ssRNA genome, extremely stable and transmitted mechanically (by contact). Besides TMV, this includes Tomato mosaic virus (Agrios, 2005).
• Cucumovirus: Genus of the family Bromoviridae. Name from Cucumber mosaic virus. These are isometric viruses with a segmented (+)ssRNA genome, transmitted by aphids in a nonpersistent manner (Blancard, 2012).
• Closterovirus: Genus of the family Closteroviridae. The name comes from Greek "kloster" — thread, due to very long, flexible filamentous particles. It includes Citrus tristeza virus and Beet yellows virus. They are transmitted by various insects, most often aphids, whiteflies, or mealybugs (Agrios, 2005).

Problems: rapid evolution, recombination, quasispecies

Virus classification is not a static system but a dynamically developing field. ICTV constantly faces serious problems related to the very biology of viruses:

• Rapid evolution: Viruses, especially RNA-containing ones, have a very high mutation rate due to their lack of repair systems (error correction) during replication. This leads to the rapid emergence of new strains and variants.
• Recombination: Viruses can exchange genome segments, especially during mixed infection. This can lead to the emergence of hybrid viruses with new properties, further complicating their classification.
• Quasispecies: Due to the high mutation rate, the viral population in a single plant is not one homogeneous molecule but a "cloud" of closely related but genetically distinct variants, called a quasispecies. This blurs the boundaries between "species" in the classical sense.

That is why ICTV taxonomy is based on a complex of characters, not just one gene, and is constantly revised.

Why the Baltimore classification is not a taxonomy

To conclude this chapter, it is important to clearly distinguish ICTV taxonomy from the Baltimore classification, which we will examine in detail below. David Baltimore in 1971 proposed a system based exclusively on the strategy of genome replication and the pathways of genetic information transfer (Koonin, 2021). He divided all viruses into seven groups, for example, "(+)ssRNA viruses" or "dsDNA viruses."

The Baltimore classification is a functional, not phylogenetic, system. It groups viruses by how they work, not by how related they are. For example, the group of (+)ssRNA viruses includes viruses from completely different evolutionary lineages, such as Potyvirus (filamentous) and Tobamovirus (rod-shaped), which are not close relatives. That is why the Baltimore classification is not a taxonomy in the strict sense and is not used by ICTV as the main hierarchy. It is an additional tool that helps understand the biology of a virus and choose methods for its diagnosis, but it does not replace evolutionary taxonomy.

In the next chapter, we will examine in detail each of the seven groups of the Baltimore classification in order to understand how these replication strategies work and why they are so important for practice.

Seven groups: dsDNA, ssDNA, dsRNA, (+)ssRNA, (−)ssRNA, ssRNA-RT, dsDNA-RT

Baltimore divided all known viruses into seven groups depending on the type of their genome and the way this genome is translated into mRNA. Let us consider each of them.

1. Group I: double-stranded DNA viruses (dsDNA). These are viruses whose genome is represented by double-stranded DNA. Their replication and transcription follow the same classical pathway as in cells: DNA → RNA → protein. This group includes many bacteriophages, as well as viruses infecting animals (e.g., herpesviruses) and plants. Among phytopathogens, these are primarily viruses of the family Caulimoviridae, such as Cauliflower mosaic virus (CaMV), and badnaviruses, for example, Banana streak virus (Agrios, 2005; Koonin, 2021).

2. Group II: single-stranded DNA viruses (ssDNA). The genome of these viruses is represented by single-stranded DNA. For replication and transcription, they must first synthesize a complementary strand, forming a double-stranded DNA intermediate, which is then used for mRNA synthesis. Most known ssDNA plant viruses belong to the family Geminiviridae. These are viruses with characteristic "twinned" particles, such as Beet yellows virus, African cassava mosaic virus, and Tomato yellow leaf curl virus (Agrios, 2005; Koonin, 2021).

3. Group III: double-stranded RNA viruses (dsRNA). The genome of these viruses consists of double-stranded RNA. Since cells do not have enzymes for RNA synthesis on an RNA template, these viruses must carry their own RNA-dependent RNA polymerase (RdRp) within the virion. This group includes viruses infecting fungi, plants, and animals. In plants, these are, for example, viruses of the genus Phytoreovirus (Rice dwarf virus) and Fijivirus (Rice black streaked dwarf virus) (Agrios, 2005; Koonin, 2021).

4. Group IV: viruses with positive single-stranded RNA ((+)ssRNA). This is the most numerous group of viruses, and probably the most important from the point of view of phytopathology. Their genome is single-stranded RNA of positive polarity, meaning it can be directly translated by host cell ribosomes like ordinary mRNA. The virion does not need its own enzymes for the first translation. This group includes the vast majority of plant viruses, including such important genera as Potyvirus, Tobamovirus, Cucumovirus, Closterovirus, Luteovirus, Polerovirus, and many others (Agrios, 2005; Koonin, 2021).

5. Group V: viruses with negative single-stranded RNA ((−)ssRNA). The genome of these viruses is represented by single-stranded RNA of negative polarity, which is complementary to mRNA. This means it cannot be directly translated. The virus must first synthesize a (+)RNA strand on its basis, which will serve as a template for the synthesis of viral proteins. For this, the virion must contain its own RdRp. This group includes viruses of the family Rhabdoviridae (e.g., Potato yellow dwarf virus) and Bunyaviridae (e.g., Tomato spotted wilt virus, TSWV) (Agrios, 2005; Koonin, 2021).

6. Group VI: reverse-transcribing viruses with an RNA genome (ssRNA-RT). These are viruses whose genome is represented by single-stranded RNA, but their replication passes through a stage of DNA synthesis using the enzyme reverse transcriptase (RT). Viral RNA is first reverse transcribed into DNA, which is then integrated into the host genome or used to synthesize new RNA molecules. This group includes retroviruses (e.g., HIV) and metaviruses (formerly known as Ty3/gypsy-like retrotransposons), which can behave as viruses (Koonin, 2021).

7. Group VII: reverse-transcribing viruses with a DNA genome (dsDNA-RT). The genome of these viruses is double-stranded DNA, but their replication also uses reverse transcriptase. Viral DNA is transcribed into RNA, which then serves as a template for reverse transcription into new DNA. This is an unusual pathway that does not occur in cells. This group includes viruses of the family Hepadnaviridae (e.g., Hepatitis B virus) and, importantly for us, Caulimoviridae (e.g., Cauliflower mosaic virus) (Koonin, 2021).

Convenience for practice, but does not reflect evolutionary relatedness

Why is the Baltimore classification so popular, despite not being an official taxonomy? The answer is simple: it is extremely convenient for practice. Knowing which group a virus belongs to, a researcher immediately obtains key information:

• Which enzymes to look for in the virion: For example, if you know that a virus belongs to group V ((−)ssRNA), you will look for RNA-dependent RNA polymerase in its composition, because without it it cannot begin replication. If it is a group IV ((+)ssRNA) virus, the presence of such a polymerase in the virion is not obligatory, although it may be present.
• How the virus enters the cell and what it does there: The replication strategy directly determines whether the virus will integrate into the host genome (like retroviruses) or remain in the cytoplasm (like most RNA viruses).
• How to diagnose it: For different groups of viruses, optimal diagnostic methods may differ. For example, for viruses with a DNA genome (groups I, II, VII), PCR with a DNA template can be used, while for RNA-containing ones (groups III, IV, V, VI), a preliminary reverse transcription reaction (RT-PCR) is necessary.

Example: (+)ssRNA viruses include different families not related to each other

The main drawback of the Baltimore classification as a taxonomic system is that it does not reflect evolutionary relatedness. The clearest example is group IV, (+)ssRNA viruses. It includes viruses from completely different, unrelated families. For example:

• Potyvirus (family Potyviridae) — filamentous viruses transmitted by aphids.
• Tobamovirus (family Virgaviridae) — rod-shaped viruses transmitted by contact.
• Cucumovirus (family Bromoviridae) — isometric viruses with a segmented genome, transmitted by aphids.
• Closterovirus (family Closteroviridae) — very long filamentous viruses transmitted by various insects.

All these viruses are united by only one common feature: their genome is (+)ssRNA. But evolutionarily they are so distant from each other that their unification into one group has no phylogenetic meaning. They independently acquired a similar replication strategy, but their origins are different.

Relationship to ICTV: an additional tool, not replacing taxonomy

Thus, the Baltimore classification and ICTV taxonomy are two complementary but fundamentally different tools.

• ICTV answers the question "who is this?" and "to whom is it related?" This is an evolutionary, phylogenetic system. It is important for understanding the origin of viruses, their relationships, and for developing strategies for breeding resistant varieties.
• The Baltimore classification answers the question "how does it work?" This is a functional system that describes the molecular mechanism of replication. It is indispensable for diagnostics, understanding pathogenesis, and choosing control methods.

In practical work, especially in the field of plant protection, it is necessary to master both approaches. Knowing that a virus belongs to the genus Potyvirus (ICTV), you understand that it is most likely a filamentous virus transmitted by aphids. Knowing that it belongs to group IV (+)ssRNA (Baltimore), you understand that for its diagnosis you will need a method based on RNA amplification (e.g., RT-PCR).

In the next chapter, we will examine in detail the main genera of plant viruses, their genomes, vectors, symptoms, and economic importance, combining knowledge obtained from both classification systems.

Potyvirus (family Potyviridae)

Potyvirus is the largest genus of plant viruses, including more than 90 confirmed species and about 90 putative ones. The genus name comes from Potato virus Y (PVY), which is the type species. These viruses cause colossal damage to many crops worldwide.

Genome and structure: Potyviruses have a genome in the form of a single (+)ssRNA molecule about 10 kb in size. Virions are long, flexible filamentous particles 11–15 nm in diameter and 680–900 nm in length (Agrios, 2005). A characteristic feature is the formation of cylindrical inclusions (pinwheel-like) in the cytoplasm of infected cells, which is a diagnostic sign (Agrios, 2005).

Vectors: Transmitted in a nonpersistent manner by aphids. Transmission requires the viral helper component protein (HC-Pro), which binds to the capsid protein and ensures attachment of the virus to the aphid stylet (Agrios, 2005).

Symptoms: Cause mosaic, mottling, chlorosis, deformation of leaves and fruits, necroses, dwarfism. Symptoms vary greatly depending on the virus, strain, and host plant.

Economic importance: Includes many destructive pathogens, such as:

• Potato virus Y (PVY): One of the most dangerous potato viruses, causing mosaic, tuber necrosis, and significant yield losses.
• Cucumber mosaic virus (CMV): Although CMV belongs to another genus (Cucumovirus), Potyviruses also include viruses infecting cucumbers, for example, Zucchini yellow mosaic virus (ZYMV).
• Plum pox virus (PPV): A quarantine object causing serious diseases of stone fruits (plum, peach, apricot), leading to deformation and fruit drop (Blancard, 2012).
• Sugarcane mosaic virus (SCMV) and Maize dwarf mosaic virus (MDMV).
• Bean common mosaic virus (BCMV).

Tobamovirus (family Virgaviridae)

Tobamovirus — a genus named after Tobacco mosaic virus (TMV). It is one of the most studied viruses in the history of biology.

Genome and structure: The genome is represented by a single (+)ssRNA molecule about 6.4 kb in size. Virions are rigid rod-shaped particles 18 nm in diameter and 300–310 nm in length (Agrios, 2005). Viral particles are extremely stable and can retain infectivity for many years in dry plant residues, on seeds, and even in tobacco products.

Vectors: They have no biological vectors. They spread mechanically — through contact between plants, via the hands and tools of workers, and also with seeds (Agrios, 2005).

Symptoms: Cause mosaic, mottling, leaf deformation (sometimes up to filiformity), dwarfism. Necrotic rings and internal browning may appear on fruits.

Economic importance: TMV infects tobacco, tomatoes, peppers, and many other solanaceous crops. Tomato mosaic virus (ToMV) — a very close relative of TMV, causes serious losses in greenhouse farms. Also included here is Cucumber green mottle mosaic virus (CGMMV), infecting cucurbit crops.

Cucumovirus (family Bromoviridae)

Cucumovirus — a genus whose type species is Cucumber mosaic virus (CMV). It is one of the most widespread and harmful viruses in the world.

Genome and structure: The genome is segmented and consists of three (+)ssRNA molecules. Virions are isometric particles about 29 nm in diameter (Agrios, 2005). CMV has a very broad host range, infecting more than 1,200 plant species, including vegetable, fruit, ornamental crops, and weeds.

Vectors: Transmitted by more than 80 species of aphids in a nonpersistent manner. It can also be transmitted mechanically and with seeds of some crops.

Symptoms: Causes mosaic, leaf deformation (sometimes up to filiformity or "stringiness"), plant dwarfism, necroses. White areas may appear on cucumber fruits, making them unsuitable for sale (Blancard, 2012).

Economic importance: CMV causes enormous damage to vegetable growing, especially cucumbers, tomatoes, peppers, lettuce, and many other crops. The virus also infects many flower crops.

Closterovirus (family Closteroviridae)

Closterovirus — a genus whose name comes from Greek "kloster" (thread), reflecting the morphology of virions. The type species is Beet yellows virus (BYV).

Genome and structure: These are the longest plant viruses. Their genome is one (+)ssRNA molecule 14.5–19.3 kb in size. Virions are very long, flexible filamentous particles 1350–2000 nm in length and 12 nm in diameter (Agrios, 2005). They contain a unique "tail" at one end, consisting of a minor capsid protein.

Vectors: Transmitted semipersistently by various insects: aphids, whiteflies, mealybugs, and scale insects, depending on the virus species (Agrios, 2005).

Symptoms: Cause yellows (chlorosis), leaf curling, dwarfism, phloem necroses. They often infect only the phloem, which complicates diagnosis.

Economic importance: Includes such important pathogens as:

• Citrus tristeza virus (CTV): One of the most dangerous citrus viruses, causing rapid death of trees grafted onto susceptible rootstock (e.g., sour orange). Transmitted by aphids.
• Beet yellows virus (BYV): Infects sugar beet, causing significant losses in yield and sugar content. Transmitted by aphids.
• Grapevine leafroll-associated viruses (GLRaV): Infect vineyards worldwide.

Other important genera of plant viruses

Besides those listed above, there are several other genera that deserve mention in our course.

• Luteovirus (family Luteoviridae): Name from Latin "luteus" (yellow). Type species — Barley yellow dwarf virus (BYDV). Genome — (+)ssRNA. Virions — isometric. Transmitted persistently (circulatively) by aphids. Cause yellows, dwarfism, leaf curling. Infect cereal crops, causing serious damage.
• Polerovirus (family Luteoviridae): Name from Potato leafroll virus (PLRV). Also transmitted persistently by aphids. Virions isometric. Besides PLRV, this includes Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV). Cause yellowing, leaf curling, growth inhibition.
• Geminivirus (family Geminiviridae): Name from Latin "gemini" (twins), since virions consist of two fused icosahedral particles. Genome — ssDNA (Baltimore Group II). This is the only family of plant viruses with such a genome that we will consider. Transmitted by whiteflies (genus Begomovirus) or leafhoppers (genera Curtovirus, Mastrevirus). Cause very serious diseases, especially in tropical and subtropical regions: Tomato yellow leaf curl virus (TYLCV), African cassava mosaic virus (ACMV), Bean golden mosaic virus (BGMV). Characteristic symptoms: leaf curling, yellow mosaic, dwarfism.
• Tospovirus (family Bunyaviridae): Name from Tomato spotted wilt virus (TSWV). This is the only group of plant viruses with a (−)ssRNA genome (Baltimore Group V) and a lipid envelope. Virions spherical, 80–110 nm in diameter. Transmitted by thrips in a persistent, propagative manner (the virus multiplies in the vector's body). Cause very dangerous diseases: spotted wilt, necroses, ring spots on leaves and fruits, bronzing. Infect tomatoes, peppers, tobacco, as well as many ornamental crops.
• Ilarvirus (family Bromoviridae): Name from "isometric labile ringspot viruses." Genome — (+)ssRNA, segmented. Virions isometric but often pleomorphic (variable in shape). Transmitted by pollen and seeds, and by some thrips species. Cause ring spots, mosaic, necroses on stone and pome fruits, roses, hops. A typical representative is Prunus necrotic ringspot virus (PNRSV).

In the next chapter, we will move on to a completely special group of pathogens — viroids, which are even smaller than viruses and have no protein coat.

Discovery: PSTVd (potato spindle tuber viroid)

The history of the discovery of viroids is a classic example of scientific curiosity. In 1971, American phytopathologist Theodor Diener was studying the causative agent of potato spindle tuber disease. He found that infectivity persisted even after treating the preparation with enzymes that destroy proteins and after phenol extraction, which removes proteins. Moreover, the agent was so small that it passed through filters that retain all known viruses. Diener concluded that the causative agent was not a virus but a free, low-molecular-weight RNA, which he called a viroid (from "virus" and "oid" — similar) (Agrios, 2005). Thus was discovered Potato spindle tuber viroid (PSTVd) — the first representative of this group.

Structure

Viroids are single-stranded, circular RNA molecules only 250 to 400 nucleotides in length. For comparison: the genome of the smallest virus is about 1,700 nucleotides long (Agrios, 2005). Due to such a short length, viroids do not encode any proteins. All their "information" is the nucleotide sequence, which determines their ability to replicate and their pathogenicity.

The viroid RNA molecule has a rod-like structure, which is formed by base pairing between complementary regions of the same strand. This structure makes viroid RNA very stable and resistant to enzymes (ribonucleases) and high temperatures (Agrios, 2005; Blancard, 2012).

Several functional domains are distinguished in the viroid structure (Blancard, 2012):

• Central conserved region: The most similar among all viroids.
• Terminal regions (left and right): Responsible for replication.
• Pathogenic region: Determines the severity of symptoms. A change of just one or two nucleotides in this region can turn a mild strain into a highly virulent one.
• Variable region: The least conserved.

Replication: via host RNA polymerase

Since viroids do not encode their own enzymes, host RNA polymerase II — an enzyme that normally synthesizes mRNA on a DNA template — is used for their replication (Agrios, 2005). This is a unique case when an enzyme intended to work with DNA is "tricked" into working with an RNA template.

The process of viroid replication occurs by the rolling circle mechanism and includes the following stages (Agrios, 2005):

1. Synthesis of (−) strand: Host RNA polymerase synthesizes a complementary (−) RNA strand using the original (+) viroid strand as a template. In this process, a long, multimeric linear (−) strand is formed.

2. Synthesis of (+) strand: The multimeric (−) strand serves as a template for the synthesis of a multimeric (+) RNA strand.

3. Cleavage and ligation: Specific host enzymes cut the multimeric strands into monomers (single viroid copies) and circularize them.

Interestingly, viroids replicate in different cell compartments depending on the family: Pospiviroidae — in the nucleus (nucleolus), and Avsunviroidae — in chloroplasts (Koonin, 2021).

Systematics: families Pospiviroidae, Avsunviroidae

The modern taxonomy of viroids, according to ICTV, is based on genome structure and replication mode. Two families are distinguished (King et al., 2012; Blancard, 2012):

1. Family Pospiviroidae: This family includes most known viroids, including PSTVd. They are characterized by the presence of a central conserved region and replicate in the nucleus. The name comes from the type genus Pospiviroid (Potato spindle tuber viroid).

2. Family Avsunviroidae: A small group of viroids that do not have a central conserved region and possess the ability to self-cleavage (ribozyme activity). They replicate in chloroplasts. The type genus is Avsunviroid (Avocado sunblotch viroid).

Genera: Pospiviroid, Hostuviroid, Cocadviroid, Apscaviroid, Coleviroid

Within the family Pospiviroidae, several genera are distinguished, whose names reflect either the host or the symptom (Blancard, 2012):

• Pospiviroid: Type genus, including PSTVd, as well as Citrus exocortis viroid (CEVd) and Chrysanthemum stunt viroid (CSVd).
• Hostuviroid: Name from Hop stunt viroid.
• Cocadviroid: Name from Coconut cadang-cadang viroid.
• Apscaviroid: Name from Apple scar skin viroid.
• Coleviroid: Name from Coleus blumei viroid.

In the family Avsunviroidae, the genera Avsunviroid (Avocado sunblotch viroid), Pelamoviroid (Chrysanthemum chlorotic mottle viroid), and Elaviroid (Peach latent mosaic viroid) are distinguished.

Diseases: potato spindle tuber, citrus exocortis, chrysanthemum stunt

Viroids cause a number of economically significant diseases. Let us consider the most important ones.

• Potato spindle tuber (PSTVd): Infects potato and tomato. In potato, plants become erect, spindle-shaped, leaves small, tubers elongate, become spindle-shaped, and their yield drops sharply (Blancard, 2012). The viroid is transmitted mechanically during tuber cutting, as well as with pollen and seeds. This is one of the most dangerous potato diseases.
• Citrus exocortis (CEVd): Infects citrus, especially trifoliate orange and citrange. On the rootstock, characteristic scaly bark develops, leading to growth inhibition and reduced yield. The viroid is transmitted by grafting and with tools (Blancard, 2012).
• Chrysanthemum stunt (CSVd): Causes growth retardation, deformation of flowers and leaves, making flowers unsuitable for sale. Transmitted with cuttings and tools (Blancard, 2012).
• Coconut cadang-cadang (CCCVd): One of the most terrible diseases of coconut palms in the Philippines. Leads to gradual death of leaves, reduced yield, and tree death. Transmitted mechanically and possibly by pollen (Agrios, 2005).

Transmission: mechanical, seeds, pollen, vegetative material

Unlike viruses, viroids have no specific vectors (insects, nematodes, fungi). They spread by the following routes (Agrios, 2005; Blancard, 2012):

• Mechanical transmission: The most common route. Viroids are transmitted by contact between plants, through hands, tools (knives for cutting tubers, pruning shears), clothing.
• Vegetative propagation: Any planting material (tubers, cuttings, bulbs, rhizomes) taken from an infected plant will contain the viroid.
• Seeds and pollen: Many viroids are transmitted with seeds and pollen, although the transmission rate may be low.
• Other routes: In rare cases, transmission by aphids is possible (only in complex with a helper virus) and through infected soil.

Practical importance: quarantine, certification, diagnostics

Viroids have enormous practical importance for agriculture, and their accurate identification is critically important.

Quarantine: Many viroids (e.g., PSTVd, CEVd, CCCVd) are quarantine objects. Their detection in a shipment of seeds or planting material entails destruction of the entire shipment.

Certification: Programs for certification of planting material (especially potato, citrus, fruit crops) necessarily include testing for viroids.

Diagnostics: Since viroids have no protein coat, serological methods (ELISA) do not work for their detection. Main diagnostic methods:

• Biological: Inoculation of indicator plants (e.g., tomatoes for PSTVd).
• Electrophoresis: Return electrophoresis in PAGE, which allows separation of circular and linear RNAs.
• Molecular methods: RT-PCR and DNA hybridization are the most sensitive and reliable methods. For this, it is necessary to isolate RNA and perform reverse transcription followed by amplification (Blancard, 2012).

In the next chapter, we will examine in more detail the practical importance of accurate identification of viruses and viroids for diagnostics, certification, and quarantine.

Diagnostics: choice of method

The first and most obvious application of accurate identification is diagnostics. However, the choice of diagnostic method is not arbitrary. It is directly determined by the biological properties of the suspected pathogen, its titer in the plant, the presence of mixed infections, and the available resources of the laboratory (Blancard, 2012).

The main methods for diagnosing viruses and viroids can be divided into several groups.

1. Biological methods (indicator plants). A classical approach in which sap from a suspected diseased plant is inoculated onto a specific set of indicator plants. By the nature of symptoms (local necroses, mosaic, ring spots), the virus can be identified with high probability. This method is labor-intensive, requires a lot of time and greenhouse space, but remains indispensable for detecting unknown or new viruses, as well as for biological typing of strains (Agrios, 2005; Blancard, 2012).

2. Serological methods. The most common is enzyme-linked immunosorbent assay (ELISA), in particular its modifications (DAS-ELISA, indirect ELISA). The method is based on specific binding of viral protein (antigen) with antibodies. ELISA is high-throughput, relatively inexpensive, and allows testing thousands of samples. However, it does not work for viroids, which lack a protein coat, and can give false-negative results at low virus titer or high genetic variability of strains (Agrios, 2005; Blancard, 2012).

3. Molecular methods. They are based on detection of the pathogen's nucleic acid. For RNA-containing viruses and viroids, the obligatory step is reverse transcription (RT) followed by polymerase chain reaction (PCR). Methods of real-time RT-PCR (qPCR) have also been developed, which allow not only detection but also quantification of virus concentration. Loop-mediated isothermal amplification (LAMP) is another promising method that does not require complex equipment and is suitable for field conditions (Kreuze et al., 2020; Jeger et al., 2023). Molecular methods have the highest sensitivity and specificity; they are indispensable for diagnosing viroids and latent infections.

4. Sequencing. With the development of high-throughput sequencing (HTS) technologies, it became possible not only to detect known viruses but also to discover new ones, as well as to analyze quasispecies and recombinant forms. HTS is becoming the gold standard for virological screening and phylogenetic analysis (Kreuze et al., 2020; Koonin, 2021). However, its use is limited by cost and the need for bioinformatic data processing.

5. CRISPR diagnostics. In recent years, systems based on CRISPR-Cas have been actively developed, which, in combination with isothermal amplification, allow highly sensitive and specific detection of nucleic acids, including plant viruses. These technologies are still at the stage of experimental development, but in the future they may become a powerful tool for field diagnostics (Jeger et al., 2023).

Problems of diagnostics. Even with modern methods, accurate identification can be difficult due to a number of factors: low titer of the virus in the plant (especially in early stages of infection), latent infections (when the virus is present but symptoms are absent), mixed infections (one plant can be infected with several viruses simultaneously, which distorts symptoms and complicates interpretation of results), as well as high genetic variability (quasispecies and recombinants). That is why reliable identification often requires a comprehensive approach: a combination of biological, serological, and molecular methods (Blancard, 2012; Jeger et al., 2023).

Certification: planting material, seeds, clones

The second key application of accurate identification is certification. For many crops, especially vegetatively propagated ones (potato, fruit, berry, ornamental), the use of healthy planting material is the basis of yield and quality. Certification is an official procedure confirming that planting material meets certain standards of health, varietal purity, and quality (Le Hingrat and Quéré, 2023).

Certification includes several stages:

• Field inspections with visual assessment of plants for symptoms.
• Laboratory testing (ELISA, PCR) to detect latent infections.
• Post-control (growing progeny under protected conditions to check health).
• Clonal propagation of source material free from pathogens (Caliskan et al., 2023; Le Hingrat and Quéré, 2023).

For example, in potato seed production, certification begins with obtaining virus-free plants in vitro (tissue culture), then propagation in protected soil (minitubers), and only after that — field generations with strict control of infection. Accurate identification of viruses (especially Potato virus Y, Potato leafroll virus, Potato spindle tuber viroid) at each stage allows rejection of infected clones and prevention of pathogen spread (Le Hingrat and Quéré, 2023). For fruit crops such as apple, pear, plum, certification is based on indexing — grafting buds or cuttings onto indicator plants, as well as molecular testing (Blancard, 2012).

Quarantine: A1, A2 lists, import ban

The third, no less important, application of accurate identification is quarantine. Many viruses and viroids are quarantine objects, that is, their import and spread on the territory of a country or region are prohibited or strictly limited. Quarantine lists (e.g., A1 — absent from the territory, A2 — limited distribution) are formed on the basis of scientific data on harmfulness, transmission routes, and the possibility of pathogen acclimatization (Le Hingrat and Quéré, 2023; Caliskan et al., 2023).

Accurate identification allows:

• Preventing import of infected material. For example, Coconut cadang-cadang viroid, Plum pox virus, Citrus tristeza virus — all are included in quarantine lists of many countries.
• Taking measures when an outbreak is detected: destruction of plants, quarantine zone, ban on export of products.
• Issuing a phytosanitary certificate for exported products, confirming their compliance with the requirements of the importing country (Le Hingrat and Quéré, 2023).

Breeding: resistant varieties, transgenic constructs

The fourth application is breeding for resistance. Knowledge of the exact taxonomic affiliation of a virus and its biological properties (e.g., transmission mechanism, host range, variability) is necessary for the targeted creation of resistant varieties. Breeders use resistance genes, often originating from wild relatives of cultivated plants. For example, the Sw-5 gene from Lycopersicon peruvianum provides tomato resistance to Tomato spotted wilt virus (TSWV), and the Ry gene from Solanum stoloniferum provides resistance to Potato virus Y (Agrios, 2005). Accurate identification of virus strains and races allows assessment of the effectiveness of these genes and prediction of their breakdown.

In addition, genetic engineering methods allow the creation of transgenic plants expressing viral genes (e.g., the capsid protein gene) to induce pathogen-derived resistance. This approach, known as cross-protection or RNA interference, requires precise knowledge of the virus genome sequence (Agrios, 2005; Jeger et al., 2023). For example, transgenic squashes resistant to Zucchini yellow mosaic virus (ZYMV) were created precisely on the basis of the capsid protein gene sequence.

Forecasting epidemics: knowledge of vectors, reservoirs

The fifth application is forecasting epidemics. Accurate identification of the virus and its vector (aphids, thrips, whiteflies, nematodes, fungi) allows building models of infection spread and developing timely protective measures. For example, knowing that Potato virus Y is transmitted by aphids in a nonpersistent manner, while Potato leafroll virus is transmitted persistently, determines different control strategies: in the first case, insecticides are ineffective, while in the second they may be effective (Jeger et al., 2023; Kreuze et al., 2020). Knowledge of virus reservoirs (weeds, wild plants, volunteer plants) allows planning crop rotations and phytosanitary measures.

Biocontrol: viruses as agents against pests (overview)

Finally, viruses can be used not only as an object of control but also as biocontrol agents. This refers to baculoviruses, which infect insect pests and are safe for beneficial insects, animals, and humans. Preparations based on baculoviruses are used to control caterpillars of Lepidoptera (e.g., cutworms, codling moths). Accurate identification of the virus agent and its host is critically important for the effective and safe use of such biopesticides (Agrios, 2005). In addition, there is the approach of cross-protection, in which plants are inoculated with a mild strain of a virus to protect against a severe strain of the same virus. This method requires accurate identification of strains and their interaction (Agrios, 2005).

Thus, accurate identification of viruses and viroids is not an academic exercise but an urgent necessity for the entire plant protection system. It underlies diagnostics, certification, quarantine, breeding, and forecasting of epidemics. Without it, neither effective management of phytosanitary risks nor sustainable agricultural production is possible.

In the next chapter, we will examine in more detail the methods for diagnosing viruses and viroids, their capabilities and limitations.

Biological methods

Biological methods, or indexing, are historically the first approach and still have not lost their importance. It is based on inoculating sap from a suspected diseased plant onto a specific set of indicator plants. Each virus causes characteristic symptoms on certain indicators: local necroses, ring spots, mosaic, leaf deformation (Agrios, 2005; Blancard, 2012).

Advantages of the biological method:

• Universality: allows detection of unknown or new viruses for which there are no diagnostic antibodies or primers.
• Biological typing: helps differentiate virus strains by their pathogenicity and symptoms.
• Accessibility: does not require complex equipment, although it requires greenhouse space and time.

Disadvantages:

• Duration: results may appear after several weeks.
• Labor intensity: requires maintaining a collection of indicator plants.
• Subjectivity: interpretation of symptoms depends on the experience of the researcher.

The biological method remains indispensable for primary screening and study of new pathogens (Agrios, 2005).

Serological methods

Serological methods are based on the specific interaction "antigen-antibody." Viral protein (antigen) binds to antibodies obtained by immunizing animals (rabbits, mice). The most common is enzyme-linked immunosorbent assay (ELISA), in particular DAS-ELISA (double antibody sandwich ELISA) and indirect ELISA (Agrios, 2005; Blancard, 2012).

Principle of DAS-ELISA:

1. Wells of a microplate are coated with antibodies to the virus.

2. A plant sap sample is added. If the virus is present, it binds to the antibodies.

3. Antibodies to the virus conjugated with an enzyme (e.g., alkaline phosphatase) are added.

4. A substrate is added, which the enzyme converts into a colored product.

5. The intensity of color is proportional to the virus concentration.

Advantages of ELISA:

• High throughput: allows testing hundreds and thousands of samples simultaneously.
• Quantitativeness: allows estimation of virus concentration.
• Simplicity and speed: results within a few hours.
• Cost-effectiveness: relatively inexpensive method.

Disadvantages:

• Does not work for viroids: they lack a protein coat, so antibodies to them are not formed.
• False-negative results: possible at low virus titer, in early stages of infection, or with high genetic variability (quasispecies).
• Cross-reactions: possible with closely related viruses.

Serological methods remain a routine tool for mass testing, especially in certification programs (Blancard, 2012).

Molecular methods

Molecular methods are based on detection of the pathogen's nucleic acid (RNA or DNA). They have the highest sensitivity and specificity and are indispensable for diagnosing viroids and latent infections.

1. Polymerase chain reaction (PCR) and its modifications.

For RNA-containing viruses and viroids, the obligatory step is reverse transcription (RT) — synthesis of complementary DNA (cDNA) on an RNA template. Then amplification is carried out using specific primers. RT-PCR allows detection of even single molecules of viral RNA (Kreuze et al., 2020; Jeger et al., 2023).

2. Real-time PCR (qPCR).

Allows not only detection but also quantification of virus concentration in a sample. This is important for assessing viral load and predicting disease severity.

3. Loop-mediated isothermal amplification (LAMP).

This method does not require a complex thermal cycler, since the reaction proceeds at a constant temperature (about 60–65 °C). LAMP has high sensitivity and specificity, and results can be seen with the naked eye (by color change or turbidity of the solution). It is promising for field diagnostics (Jeger et al., 2023; Kreuze et al., 2020).

4. DNA hybridization.

The method is based on the ability of complementary nucleic acid strands to bind to each other. Labeled DNA probes (radioactive or chemiluminescent) hybridize with viral RNA or DNA applied to a membrane. The method is sensitive but inferior to PCR and requires special equipment (Blancard, 2012).

Advantages of molecular methods:

• Highest sensitivity: detection of single copies of the genome.
• Specificity: ability to distinguish closely related strains.
• Work with viroids: the only reliable method for their diagnosis.
• Speed: results within a few hours.

Disadvantages:

• Cost: equipment and reagents are expensive.
• False-positive results: possible when samples are contaminated.
• Need for a laboratory: most methods require an equipped laboratory and trained personnel.

Sequencing

High-throughput sequencing (HTS) is a revolutionary method that allows reading the sequence of nucleic acids in a sample without prior knowledge of the pathogen. HTS allows:

• Detecting known and unknown viruses.
• Analyzing quasispecies and recombinant forms.
• Studying phylogeny and evolution of viruses.
• Performing metagenomic analysis of entire communities (Kreuze et al., 2020; Koonin, 2021).

HTS is becoming the gold standard for virological screening, but its use is limited by cost and the need for bioinformatic data processing.

CRISPR diagnostics

CRISPR diagnostics is the newest direction, combining isothermal amplification with CRISPR-Cas systems. For example, the SHERLOCK system (Specific High-sensitivity Enzymatic Reporter unLOCKing) uses Cas13 to detect RNA, and DETECTR uses Cas12 to detect DNA. These systems have high sensitivity and specificity and can also be adapted for field conditions (Jeger et al., 2023). So far, CRISPR diagnostics is at the stage of experimental development, but in the future it may become a powerful tool for rapid identification of viruses and viroids.

Problems of diagnostics

Even with modern methods, accurate identification can be difficult due to a number of factors (Blancard, 2012; Jeger et al., 2023):

• Low titer: in early stages of infection, virus concentration may be below the detection limit of the method.
• Latent infections: the virus is present in the plant but symptoms are absent, complicating detection.
• Mixed infections: one plant can be infected with several viruses simultaneously, which distorts symptoms and can lead to cross-reactions.
• Quasispecies: high genetic variability leads to primers or antibodies not recognizing all variants.
• Inhibitors in plant sap: some components of sap can inhibit PCR or ELISA.

To overcome these problems, a comprehensive approach is used: a combination of biological, serological, and molecular methods. For example, in potato certification, visual inspections, ELISA for mass screening, and RT-PCR for confirmation and detection of latent infections are used (Le Hingrat and Quéré, 2023).