Clinical virology and antiviral resistance

Clinical virology and antiviral resistance

Virus structure

Viruses are small obligate intracellular parasites; they need a host cell to replicate their nucleic acid, produce proteins, and make energy. They must be infectious to endure in nature; if not infectious, a virus cannot survive. They contain RNA or DNA but not both. All organisms have their specific virus (bacteria, animals, humans).

Viral components are assembled and do not replicate by division. The different components are produced and then assembled together. Viruses are smaller than classical filter pores of 5 µm, so they are not separated from other bacterial cells through these filters. To be captured, they need smaller pores; initially, a filter pore of 0.3 µm was used.

Nucleic acid and capsid compose the naked viruses, while enveloped viruses are characterized by the presence of a lipidic envelope outside the capsule. Nucleic acid and capsid together form the nucleocapsid; in some cases, there are also particular enzymes.

Naked viruses are very stable; they are resistant to heat, proteases, acid, detergents, and as a consequence, they can spread very easily among their hosts. They can survive in the environment. On the other hand, the envelope is susceptible to detergents and high temperature, so these viruses cannot survive in particular environments (e.g., gastrointestinal tract). These viruses must stay wet and spread in large droplets (e.g., coronavirus).

Symmetry (how the capsomers are arranged in the capsid) is similar for enveloped and naked viruses. In some viruses, capsomers assemble in an icosahedric shape, and in others, in a helicoid shape.

Virus classification

Viruses are classified in: order (virales), family (viridae), subfamily (virinae), genus (virus), and species. There is an international committee for virus taxonomy. The advent of nucleotide sequence determination has revolutionized biology and largely rationalized taxonomy, including that of viruses.

The universal virus taxonomy provides a classification scheme that is supported by verifiable data and expert consensus. It is an indispensable framework both for further study of the currently recognized virus species and for the identification and characterization of newly emergent viruses, whether they result from natural, accidental, or deliberate dissemination.

There are several families affecting humans; Herpesviridae, Polyomaviridae, Adenoviridae, Papillomaviridae, Poxviridae (smallpox is a possible bio-weapon), Iridoviridae, Hepadnaviridae, Orthomyxoviridae, Retroviridae, Paramyxoviridae, Filoviridae, Retroviruses, Rhabdoviruses, etc.

Virus replication cycle

The virus replication cycle starts with the attachment to the host cell surface, then penetration and injection of the viral nucleic acid into the cytoplasm. Inside the cell, viral genes are transcribed, the mRNA is translated, and proteins are synthesized. In some cases, viral DNA can be incorporated into the host genome. For RNA viruses with a positive-sense genome, the RNA is directly transcribed, while for negative-sense genome, they need to synthesize a positive strand.

The growth cycle of a non-enveloped, double-stranded DNA virus involves multiple steps in the replication cycle that take place in the nucleus. After penetrating the host cell, viral DNA is uncoated and enters the nucleus. Viral genes are transcribed, and the mRNAs are translated in the cytoplasm. Newly synthesized proteins enter the nucleus. Viral DNA is replicated in the nucleus, sometimes with the help of newly synthesized viral replication proteins. Viral DNA and viral structural proteins assemble in the nucleus to produce new progeny virions. On rare occasions, viral DNA may be incorporated into cellular DNA as a side effect of infection.

The growth cycle of a positive-sense, single-stranded RNA virus occurs in the cytoplasm. The virus enters the cell, and the viral RNA genome is uncoated. As a positive-sense, single-stranded genome, the RNA is directly translated, producing viral proteins. A negative-sense RNA copy of the positive template is synthesized. It is used to produce many positive-sense copies. The newly synthesized positive-sense RNA molecules are assembled with viral structural proteins to produce new progeny virions.

• Class 1: ds DNA viruses behaving like our cells
• Class 2: ss DNA viruses
• Class 3: ds RNA Rehoviridae
• Class 4: ss RNA +
• Class 5: ss RNA –
• Class 6: ss RNA + retroviruses, the RNA doesn’t work as a messenger

Viruses provide essential enzymes for the biotechnologist, such as DNA-dependent RNA polymerase, DNA ligase, RNA ligase, and reverse transcriptase (most of them are now engineered derivatives with improved features such as processivity and thermostability). Human viruses contain few genes. The mutation rate is different among viruses, from 10^-2 for viroids to 10^-9 for dsDNA viruses (without proofreading activity). In eukaryotes, this rate is about 10^-11, so our genome is highly stable.

The larger the genome size, the lower is the mutation rate. In evolution, viruses have a bigger advantage than us because we have a more stable genome with lower mutation rates (meaning slower evolution). An excessive mutational load in large genomes (like our genome) leads to genetic meltdown. Too many mutations in viruses mean viral extinction because the virus loses the ability to correctly replicate, on the other hand, few mutations lead to clearance by immune response. Many viruses have accommodated their mutation rate in a safe zone that allows them to replicate and evolve. The coronavirus is very stable; it is the only virus with proofreading activity (evolutionary stable virus), this leads to important consequences such as vaccines and the effective immune response.

Variability and viral evolution

Variability is the key to exploring the genetic landscape of viruses and microorganisms. Replication errors are the major component of variability; no errors mean no evolution. However, there is a price to pay: many variants are unfit or non-viable, some errors lead to non-viable organisms and genetic imbalance.

Recombination is one mechanism through which a new genome is assembled starting from other different genomes. The two genomes break, and then there is a ligation between them, leading to the production of a new genome (non-replicative recombination), typical of DNA viruses. Template switching or replicative recombination is another kind of recombination; in this case, polymerase falls off templates and binds another template to the first one, typical of RNA viruses and retroviruses. Polymerase changes template during the process of replicating.

Transduction relies on integration into the host genome, and during the excision, host genes are acquired in the viral genome. This process is common among retroviruses and bacteriophages. Acutely transforming retroviruses (only for animals) are characterized by this kind of recombination.

The last recombination is the reassortment (characteristic of influenza virus), the genome is composed of different segments that replicate and can be mixed and packaged in the same capsid. Influenza viruses combine template switching and reassortment. We call antigenic drift the mechanism through which each year influenza mutates, and this is the reason why every year we need new vaccines. Influenza can also make a complete mutation by antigenic shift, a process by which totally new variants are produced. It can be a consequence of reassortment in animals (birds and swine) considered mixing vessels.

Influenza makes small errors—mutations—when it copies its genetic code during reproduction. But influenza lacks the ability to repair those errors because it is an RNA virus; RNA, unlike DNA, lacks a self-correcting mechanism (note that coronaviruses are an exception to this rule). As a result, influenza is not genetically stable. Every generation is slightly different, and those differences accumulate as time passes. That slow deviation is called antigenic drift, and it is the reason why it is necessary to reformulate flu vaccines every year.

Every flu season, the genetic makeup of the dominant strains from the prior year will have drifted, changing the surface structure of those strains just enough to diminish, or even destroy, the effectiveness of the previous year’s vaccine. Each winter, health authorities must make an educated guess about which strains are likely to dominate in the next flu season. It takes six more months to develop and manufacture vaccines for chosen strains. But in some years, genetic drift during just those six months can render the newly formulated vaccine ineffective, leaving populations more vulnerable to the newly evolved virus.

Rapid change in circulating flu viruses is known as antigenic shift. Shift can happen in several ways: when a flu strain jumps to a species which it has never before infected, or when the process of viral reproduction creates a novel strain. In either case, the human immune system perceives the strain as unfamiliar. That lack of immunologic experience is what causes the much larger amounts of illness that are created by a shifted strain.

Antigenic shift occurred in 1997 when the H5N1 avian flu virus appeared for the first time in humans, one example of what happens when a flu strain jumps from one animal species to another, or from an animal species to a human host. However, the H5N1 virus has so far failed to spread from human to human due to insufficient adaptation. Antigenic shift can also occur when at least two influenza A strains infect animals simultaneously and combine to form a new virus that contains a mixture of the genes of the two original strains (reassortment). This can also happen in people infected with two strains at the same time, although birds and swine are considered the major "mixing vessels”.

DNA viruses use our cells for integrating their genome; they are characterized by latency and persistence in the host (e.g., herpes simplex, cytomegalovirus, varicella-zoster). Our cells are designed to accommodate DNA, and unfortunately, this characteristic is used by DNA viruses. DNA viruses are able to infect cells and persist in them. They can be latent but can also reactivate and stimulate our immune system, but they can also hide from it and from drug pressure. Latency is the key strategy for persistent infection.

RNA viruses cannot hide in our cells because our RNA is continuously destroyed, so they replicate very fast. In this case, viruses cannot hide in the cell, and they rely on error-prone replication by which they can generate multiple variants. There is an exception, which is the hepatitis C virus, but we have drugs to eradicate it.

Retroviruses are the cleverest solution that nature has prepared in this contest. They have an RNA genome in the viral particle and an error-prone replication, but their genome is converted into DNA and is integrated into the host genome. They combine previous features, making it difficult to eradicate them. HIV is the most important example; it can generate variants continuously, so a vaccine is not available. We have efficient drugs, but we cannot eradicate it.

Quasispecies: a distribution of mutants that is generated by a defined mutation-selection process. Selection among these species is provided by our immune system. In HIV and HCV, each replication creates a different variant, so there are a number of related new genomes that constitute a quasispecies. Each variant is tested by our immune response that can completely eliminate it; other variants aren’t recognized and persist in the host (intrahost evolution).

Consequences of viral evolution

• Emerging viruses are viruses able to cross the human species barrier. Some of them jump from animals to humans but without human-to-human transmission, whereas others with human-to-human transmission like SARS-CoV-2.
• Some viruses can escape immune response and antiviral drugs.
• Mutation can abolish the recognition between antibodies and viral epitopes.
• Insufficient protection (HIV), and the need to reshape vaccines (influenza).

Bats can carry many kinds of viruses without becoming ill in response to them. There is a huge natural coronavirus pool in bats that sometimes spreads to humans. For example, the Ebola virus originated from the Angora dog bat (Mops condylurus, a fruit-eating bat), although its intermediate host is still unknown. The MERS (Middle East Respiratory Syndrome) virus originated from the Egyptian tomb bat (Taphozous perforatus) and was transmitted to the dromedary camel (Camelus dromedarius) before going on to infect humans.

Immune response to viral infections

Viruses are very good antigens; antigens are generally viral proteins that present a number of different antigenic determinants called epitopes. Each epitope can be detected by our immune system, and if they mutate or change, our immune systems will not be able to recognize them anymore.

Virus polymorphism translates into variation in the different epitopes. Our immune system is also very polymorphic, meaning that antibodies one person generates against a pathogen are different from antibodies produced by other people. There is not a pathogen able to kill all people. The interaction of the virus with the immune system results in different ways, from a mild infection to a severe one.

Innate immunity and adaptive immunity are our defenses against pathogens. The first one recognizes non-specific pathogens and acts in the same way for all of them, whereas adaptive immunity is specific for a single pathogen and is characterized by memory. Innate immunity is the first line of defense, whereas the adaptive is the second line of defense. The innate immunity limits the initial spread of the virus and triggers adaptive immunity.

Adaptive immunity cells are lymphocyte T and B, and it is characterized by memory. Innate immunity is an essential step because, through PAMPs (Pathogen-Associated Molecular Patterns), a pathogen is recognized and detected by macrophages and other immune cells that have pattern recognition receptors (PRRs). The interaction between PAMP and PRR allows phagocytosis and the destruction of the pathogen, causing the release of cytokines that attract other cells and trigger adaptive immunity.

Adaptive immunity has high specificity but is not preformed in amounts sufficient to control infection. It needs some time to develop but generates a memory status allowing a more effective and prompt response to future infections by the same agent. In adaptive immunity, each lymphocyte T or B is specific for only one pathogen.

Antibodies are produced by B lymphocytes that have been sensitized by the antigen and differentiated into plasma cells. Antibodies travel through our body and inactivate the pathogen. T lymphocytes meet APC (antigen-presenting cells) that sensitize them, inducing differentiation into effector T cells.

The innate immunity is based on phagocytic cells, natural killer (NK) cells, and the complement system. It is very fast. There are several molecules that can interfere with incoming invaders such as viruses and bacteria, some of which are specific for a particular kind of pathogen (gram-positive or gram-negative bacteria; toll-like receptors specific for dsRNA, ssRNA, etc.).

Interferon is a system of related proteins that can be activated by viral infection. Before dying, an infected cell synthesizes interferon (alpha and beta) that are able to find receptors on uninfected cells and communicate the viral presence. The interferon binds to a cell surface receptor on other cells and induces the production of antiviral enzymes (antiviral state). After the binding, uninfected cells can activate a series of systems to defend themselves and other related cells. Interferon does not protect the initial cell, but protects the other cells by inducing the production of antiviral enzymes.

Natural killer cells have a couple of key receptors; one controls other cells’ MHC1 and the other interacts with cell stress proteins on tumor cells or pathogen-infected cells. A cell in a normal state presents a lot of MHC1; a viral or a bacterial infection downregulates the expression of these receptors and increases the exposure of stress proteins, allowing NK cells to recognize those cells as infected. After recognition, NK cells bind to the infected cell and release perforins and granzymes that induce cell lysis.

In viral infections, the most important immune cells are lymphocytes T and B, NK cells, dendritic cells, and macrophages. Adaptive immunity contains cell-mediated immunity (specialized cells for antigen recognition, cellular cross-talk, and elimination of virus-infected cells) and humoral immunity (generation and secretion of antibody to neutralize viral particles and avoid infection of new cells). Cellular cross-talk is mediated by molecules and surface structures.

Recap of MHC1 and MHC2, immunoglobulins, antigen presentation

Class I MHC molecules are found on all nucleated cells and are the major determinant of "self." Each cell expresses a pair of different HLA-A, HLA-B, and HLA-C proteins, one from each parent, providing six different clefts to capture a repertoire of antigenic peptides. The class I MHC molecule presents antigenic peptides, most of which are from within the cell (endogenous), to CD8-expressing T cells. When the virus infects the cell, it causes the production of its viral protein that is recognized and becomes a target for ubiquitin. They are delivered to the proteasome, which digests them into peptides that are transported to the endoplasmic reticulum where they are conjugated to MHC1. The complex is translocated to the surface of the cell. Now this cell becomes a target for the immune system.

Class II MHC molecules are normally expressed on antigen-presenting cells, cells that interact with CD4 T cells (e.g., macrophages, DCs, B cells). The class II MHC molecules are encoded by the DP, DQ, and DR loci and, like MHC I, are also co-dominantly expressed to produce six different molecules. The class II MHC molecule presents ingested (exogenous) antigenic peptides to CD4-expressing T cells. Class II MHC molecules assemble in the ER with an invariant chain protein to prevent acquisition of a peptide in the ER. They are transported in a vesicle through the Golgi apparatus. Exogenous antigen (phagocytosed) is degraded in lysosomes, which then fuse with a vesicle containing the class II MHC molecules. The invariant chain is degraded and displaced by peptides of 11 to 13 amino acids, which bind to the class II MHC molecule. The complex is then delivered to the cell surface for presentation to CD4 T cells.

TH1 cells (T-inflammatory cells) are activated by antigens presented on macrophages in the context of MHC II protein.