Medical Virology PDF

DATE: sbob #1 - 02/12/2021 COURSE: Biopathology I PROFESSOR: Santangelo MODULE: Medical virology TOPIC: Medical virology introduction BOOK REFERENCE: page # SBOBINATORS: D’Amato, Grassia REVIEWER:...

DATE: sbob #1 - 02/12/2021 COURSE: Biopathology I PROFESSOR: Santangelo MODULE: Medical virology TOPIC: Medical virology introduction BOOK REFERENCE: page # SBOBINATORS: D’Amato, Grassia REVIEWER: done bb Medical Virology Why do we study viruses? Viruses are everywhere They can infect all living organisms, from bacteria to plants and human. They have a very strong specificity to the type of cell that hay can infect We carry viral genomes as part of our own genetic material Viruses can be beneficial having particular interactions with some organs in the body Mainly viruses can cause human diseases Viruses can infect different organisms, not only humans but also bacteria: this is very important for the mechanism of genetic rearrangement and diversity. The pathogenicity of viruses is based on their capability to interact with the host, in fact after having infected a part of the body the virus can survive there for the rest of its life. Different diagnoses are based on the origin of the viral infection and the site of action. Sometimes viruses can be beneficial mainly in marine ecology, where the viral particles are the most abundant biological entities, in fact they are 15 times more abundant than Bacteria and Archaea. Viral infections in the ocean kill 20% to 40% of marine microbes daily, converting these living organisms into phytoplankton. The Virome Virtually every part of our body is susceptible to viral infection, that can infect mostly all of the cells of the body. When the virus enters in contact with the host it can persist inside in either an active or latent form, in fact a lot can cause infections but not real diseases, causing indeed, an asymptomatic infection in an immunocompetent host while a symptomatic one in an immunocompromised host. Surprisingly, the vast majority of the viruses that infect us have little or no impact on our health or wellbeing, because a lot of time the virus persists in a latent form inside the cell History of virology - How old are viruses? Study of molecular evolution place some viruses among the dinosaurs, although viruses have been known as distinct biological entities for little more than 100 years, around the end of 19th century, likely originated billions of years ago. First vaccine - Variolation The first vaccine is dated in the 11th century and was the first step for vaccination: it was the inoculation of healthy individuals with pus from a smallpox pustule. Consequently, there was the production of specific antibodies against the virus to prevent and contrast the infection. Thanks to this we had the knowledge that also a small particle can cause a big disease such as smallpox. 1  Another important discovery is related to Tobacco virus: Ivanovsky and Beijerinck observed that the causative agent of tobacco mosaic disease was an infectious liquid (contagium vivum fluidum) that was not retained by the filters used to remove bacteria from extract: it is composed of such small particles that can pass the normal sterilized filter, in that way scientists can have them in different liquids or environment. In 1898, Loeffler and Frosh observed that the agent of foot and mouth disease was filtrable, this led to the conclusion that viruses are infectious filtrable agents composed of small particles. Another important characteristic of viruses is that they need a living host cell in order to replicate, if this doesn’t happen no infection occurs. Differently, bacteria can growth in synthetic or any other type of media while viruses need a host living cell in order to replicate and provoke an infection. The failure of both pathogens to multiply in solutions that readily supported the growth of bacteria, as well as their dependence on host organisms for reproduction, further distinguished these new agents from pathogenic bacteria: the latter can grow in broth, in contrast respect the viruses, that need a loving host Figure 1: This graph shows the discoveries of the different pathogens; the study of viruses started with the isolation in 1935 of influenza virus. The influenza virus (one of the most common) from April 2020 to May 2021, due to the presence of the COVID, did not show an high prevalence. 2 What is a virus? “it is a piece of bad news wrapped in a protein coat.” D.Baltimore A virus is like a poison A virus is an infectious, obligate intracellular parasite (it needs a living host cell to initiate the infection and reproduction) comprising genetic material (DNA or RNA), surrounded by a protein coat and/or an envelope derived from a host cell membrane. Consequently, a virus is formed by:  a genome that can either be DNA or RNA: it is NEVER possible to find both inside the cell as part of the genome, only one of them can form the genome. We could find RNA in a DNA-virus, but RNA in this case is not part of the genome, it could be mRNA or intermediate RNA  a protein coat, around the genome  only some type of viruses presents also an envelope derived from the host cell membrane with specific glycoproteins on the surface that are typical of the virus. It covers the capsid (protein coat) of the virus. Viruses are usually small (from 20 to 300 nm of diameter): 20 nm size is represented by parvovirus and the 300nm size is represented by the smallpox virus. It is possible to see virus through electron microscope Viruses can exist in two phases: 1. when they are outside the cell/host, viruses are defined as virions while, 2. when they are inside the cell/host, in the infected cell, where they able to reproduce and star the life cycle. Viral pathogenesis is based on the interaction with the host and its reproduction within it: in fact the virus is an obligate intracellular parasite and cannot exist in this particular form outside the host. Differently from them, fungal and bacterial pathogenesis is based on the production of toxins or release of proteins that elicit an immune response. The Virion The virion is the real viral and infectious particle. A virion consists of either DNA or RNA (constituting the genome) usually complexed with protein into a core, surrounded by a protein coat called the capsid, altogether called nucleocapsid. The capsid is composed of identical subunits called capsomeres. 3 Definitions Virion: physical particle of the virus Core: nucleic acid and tightly associated proteins within the virion, that stabilize the genome Capsid: protein shell around nucleic acid or core Capsomere: protein subunit making up the capsid Nucleocapsid: core and capsid Envelope: lipid membrane with specific viral proteins found on top or within the membrane, often derived by budding from infected cell. The envelope is not common to all viruses but only in some types have it. Virus taxonomy Generally, for the viruses nomenclature, we can distinguish: → Orders: names end with -virales (ex: herpesvirales) → Families: names end with -viridae (ex: Herpesviridae) → Subfamilies: names end with -virinae (ex: alpha-beta- gamma-herpesvirinae) → Genera: common name we use for the virus (ex: simplexvirus) The International committee on taxonomy of virus classified the known families of viruses based on their structure, genome, mechanism of infection, replication and pathogenesis. They are distinguished based on different characteristics:  The structural characteristics taken in consideration are:  Symmetry of protein shell (the capsid)  Presence or absence of lipid membrane (the envelope)  The genomic characteristics taken in consideration are:  Genome architecture  Nature and sequence of nucleic acid in virion  Virus replication: depending on the type of genome viruses undergo different type of replications  Physical and biologic properties 4 Figure 2: virus classification Viruses can be divided (fig.2) according to:  Type of nucleic acid: o DNA o RNA  Symmetry of the capsid: o RNA viruses: icosahedral or helical o DNA viruses: icosahedral, helical or complex  Envelope presence o naked o enveloped  Genome architecture: o positive o negative o single (ss) o double stranded (ds) 5 Structure of viruses 1. Genetic material: core of RNA or DNA that contains the sufficient genetic information for the virus to copy itself 2. Protein shell: the shell known as the capsid, surrounds the genetic material of the virus. Capsids have geometric shapes 3. Capsomer: the protein shell (capsid) is made of many subunits called capsomers 4. Outer envelope: some viruses have a protective outer layer membrane that is acquired from the cells they have infected 5. Surface protein: proteins on the surface attach to specific receptors on host cells. Viruses without envelopes also have these proteins. Proteins can either be transmembrane or outer membrane. They are fundamental for the linkage of the virus to the host cell Functions of structural proteins The most important functions are the protection of the genome and the delivery of it. In order to protect the genome in the most appropriate way structural proteins o assemble in a stable, protective protein shell, o present specific recognition patterns to pack around the nucleic acid genome o and interact with the host cell membrane in order to form an envelope. While the delivery of genome is driven by structural proteins that o bind to the host cell receptors, o uncoat the genome, o fuse with the cell membranes o and transport the genome to the appropriate site in order to have replication. Virus particles are metastable, coswquenlty:  They must protect the genome and give it the stability,  They must come apart on infection when they are unstable and provide the cell- to-cell surface interaction to permit the virus to enter the host cell. 6  How is metastability achieved? The virus can be found in two different structures: Stable structure that is created by symmetrical arrangement of many identical proteins to provide maximal contact. The virus is usually found in this form when free in the environment. Unstable structure that is usually found when the virus is inside the host cell and it is not permanently bonded together and can be taken apart infection to release or expose the genome. The instability is necessary within the host cell in order to let the virus perform all the pathogenic reactions. Symmetry of protein shell: the capsid The capsid can either be helical or icosahedral and can be studied by transmission electron microscopy (EM), cryo electron microscopy and x-ra diffraction (most used). Examples: 1. HELICOIDAL o Ebola Virus: filovirus 2. ICOSAEDRAL o Herpes virus, influenza virus Helical Symmetry The capsid is composed of multiple copies of a single kind of protein subunit arranged in a close-packed helix, which places every subunit in the same microenvironment (fig 3) Figure 3 Each subunit has identical bonding contacts with its neighbours. Capsid protein subunits+nucleic acid will self-assemble in vitro in an energy- independent fashion Self-assembly also occurs in the absence of RNA/DNA. Nucleocapsids of influenza, measles, mumps, rabies, and poxviruses are constructed with a helical arrangement of protein. 7 Icosahedral Viruses: The construction of a spherically (icosahedral) shaped virus involves the packing together of many identical subunits, placed resembling an icosahedron: 12 vertices, 30 sides, and 20 triangular faces. At the electron microscope, many naked capsid viruses appear as spherical particles with a surface constructed of identical ball-shaped subunits (which are the capsomeres). Capsomeres are surface structures composed of five or six protein molecules. In the simplest virus with cubic symmetry, five protomers are placed at each one of the 12 vertices of the icosahedron to form a capsomere called a pentamer. We have protomers giving us the face and the pentamers giving us the spikes. This structure possesses different axes of symmetry, this is important for having stability of the capsule structure. Special surface structures Many viruses have structures that protrude from the surface of the virion. In virtually every case, these structures are important for the two earliest steps of infection—adsorption (linkage to the viral anti-receptor to the cell receptor) and penetration (proteins able to stabilize the fusion of the membranes when the virus enters in the host cell). We classify viruses based on their coating: naked or enveloped virus. In the naked conformation, the surface structures are in the capsid; but if the envelope is present, the surface structures are found within it We can classify the virus as (fig 4):  Naked capsid viruses, that have a nuclei acid genome within a protein shell No lipoprotein envelope. 8   Enveloped viruses that have a nucleocapsid package into a lipoprotein envelope. Nucleic acids + capsid + envelop. Figure 4: naked vs enveloped vrus Envelope: Some viruses have additional lipoprotein envelope, composed of virally coded proteins and host lipids. Enveloped viruses are readily infectious only if the envelope is intactbecause only when the anti-receptor has the proper structure it can interact with the cellular receptor, giving a functional linkage Agents that damage the envelope, such as alcohols and detergents, reduce infectivity. That’s because they destroy the anti-receptors in the envelop. Enveloped viruses obtain their envelope by budding through a host cell membrane. The viral envelope is covered with glycoprotein spikes Spikes are virus-specific which can firmly interact with receptors in the cell membrane allowing the penetration inside the cell. 9 Last year question: Don’t viruses produce the envelope inside the cell? - Generally, once the different parts of the virus, capsid and genetic materials are produced, these parts get to the site where viral glycoproteins are, but the mechanism of escaping varies. (unclear answer) Genome structure The genomes of viruses can be either RNA or DNA, but not both DNA or RNA genomes may be single- or double-stranded Genomes may be linear or circular Some genomes are segmented Some genomes are incomplete The nature of genomes is diverse, we may find single or double stranded DNAs\RNAs, length also varies as it happens with spatial arrangements, genome can be segmented or circular. Figure 5: genome charcateristics A particular class of virus is the ssRNA belonging to RNA DNA viral class are retroviruses having single stranded RNA with a retrotrascriptase enzyme inside. dsDna of Hepadnaviruses need to make RNA intermediates first. Afterwards, a retrotrascriptase gives us the final viral genome. Viral DNA or RNA genomes are structurally diverse We can observe different genomes: Linear Circular Segmented Gapped 10  ssRNA(+)generate directly a mRNA and immediately start proteim synthesis ssRNA(-) Single stranded ambisense(+/-) genomes have a negative and a positive portion linked together with a loop. Double strands can covalently attach to proteins Cross-linked ends of dsDNA DNA with covalent attached RNA Last year question: What’s the actual meaning of positive\negative strands? A: The positive one resembles mRNA, it’s immediately transcribed and traduced in a different protein, the negative one is complementary to the positive one and needs to become positive before starting the real replicative cycle. X: Difference between gapped and linear? A: Regarding the gapped type, it’s a partial double stranded circle and not a complete one. It’s a circular, but partial. These viruses need to complete the circle first and then can start replicating, using intermediate DNA forms. RNA VIRUSES: -Among Flaviviridae is Hepatitis C virus. -Among Togaviridae is Rubella virus. -Among Retroviridae is HIV. -SARS-CoV2 is a Coronaviridae. -Hepatitis A virus and enteroviruses are Picornaviridae (the smallest for this type of RNA virus). -Influenza virus is a Orthomyxoviridae. -Parainfluenza and Measles morbillivirus are Paramyxoviridae. -Rabies virus is a Rhabdoviridae. -Ebola virus is a Filoviridae -Bunyaviridae and Arenaviridae can cause hemorragic fever. 11 DNA VIRUSES: -Parvoviridae: Parvovirus B19, smallest DNA virus; -Papillomarividae: Human Papilloma virus (HPV); -Polyomaviridae: JC virus; -Adenoviridae: Adenovirus1; -Herpesviridae: big family of DNA viruses; -Hepadnaviridae: Hepatitis B virus; -Poxviridae: the agent of smallpox. 12 Baltimore Classification: (wiki) a virus classification system that groups viruses into families, depending on their type of genome (DNA, RNA, single-stranded (ss), double-stranded (ds), etc..) and their method of replication. (she read the image for the different classes) Viral Replication Viruses are obligate intracellular parasites. The viral replication can be divided in different stages:  Stage 1: The virus needs to infect a cell. Once the virus reaches a vulnerable host cell, it attaches using surface proteins. The host cell has a surface protein that acts as a Receptor and is usually an important structure for the life cycle of the cell; the virus has an Antireceptor, the structure recognized by the host.  Stage 2: After the Receptor-Antireceptor interaction, the virus’ genetic material goes inside the host cell, and regular cell life cycle is interrupted.  Stage 3: Viral DNA or RNA takes over the host cell machinery, copying itself.  Stage 4: After the virus has replicated, the new viral particles break off from the host cell and go infecting other cells. If viral particles are too large to diffuse across the plasma membrane, they can either lyse the cell releasing the content outside or proceed through gemmation. 13 General phases in animal virus multiplication cycle: 1. Adsorption - binding of virus to specific molecule on host cell 2. Penetration – virion and/or genome enters in host cell 3. Uncoating – the viral nucleic acid is released from the capsid 4. Synthesis – viral components are produced 5. Assembly – new viral particles are constructed 6. Release – assembled viruses are released by budding (exocytosis) or cell lysis, the virus makes an envelop using the cell membrane. (HIV can be released in both ways) e.g. In case of influenza virus, the receptor is the heparan sulphate, present in our cells’ membranes in the respiratory apparatus. Generally, the receptor is a protein from our cell membranes, bound by the virus to access. 1- Attachment: Attachment is one of the most important pathogenic mechanisms for a virus, only sensitive cells can interact with the virus linking it. Absorption involves attachment of a viral surface protein or spike to the cell surface receptor protein. Sometimes one linkage is enough, in other cases instead, we also need a coreceptor. Different interactions may be needed to have an infection. The mechanism of attachment varies for naked or enveloped viral species: -Enveloped: fusion of the envelop with the cell membrane and the viral particles are released; endocytosis can also occur, in which host cell membrane takes up the viral particles, and then releases them. -Naked: Direct pore mediated penetration. About protein -protein Linkage: 14 e.g. Regarding influenza virus the linkage with heparan sulphate on cell surface is enough. HIV, instead, needs a first attachment of gp120 to cd4 and after we need the 3 rd loop of gp120 binding to a coreceptor (generally CCR5 or CXCR4 depending on weather we have a macrophage or a leukocyte). Only when the double attachment is completed the virus can fuse and penetrate. Absorption of a naked virus (like in the case of rhinovirus, adenovirus or papilloma viruses) occurs generally through passage in a channel like way. 2- Penetration: We can have endocytosis, generally for enveloped ones but it can happen also with some naked viruses. The virus binds the receptor, stimulates the cell to endocytose and modification of the different viral glycoproteins occurs (like for influenza virus). The penetration by diffusion is also generally made by enveloped species, releasing the capsid inside the cell. 3- Uncoating: Uncoating occurs in both cases, we have the degradation of the capsid, and the nucleic acids are then free inside the cell. 4- Synthesis and replication -Generally, for DNA viruses the genome arrives in the nucleus for replication and transcription (except poxviruses). -RNA based viruses are generally replicated directly in the cytoplasm and never reach the nucleus (except retroviruses). All they need is present in the cytoplasm. The common point in the different mechanisms of replication is that all viruses must make RNAs able to produce typical viral proteins. David Baltimore (boy from the Baltimore classification) says that viral genomes must make RNAs readable from hosts’ ribosomes. That’s the common point. Genomes that are directly mRNAs (single stranded positive) are immediately transcribed. Here are the mechanisms of replication for each class. She read these examples. 15 Q: If we have an RNA (+), why is it transformed first in Rna (-) and then again into a positive stranded Form? A: Because usually they need to form intermediates to duplicate the genome, in the different cycles we want different lines of replications, on one side we have the duplication of the new viral genome, from the other side we have the transcription of the different part of the genome. A parallel path, more direct. Class I _ dsDna These types of viruses usually must enter the host nucleus before it is able to replicate. Furthermore, these viruses require host cell polymerases to replicate the viral genome and, hence, are highly dependent on the cell cycle. mRNA is transcribed in the normal way from viral DNA using the host transcriptase enzymes, into two types of mRNA's: 1) early mRNA, transcribed prior to the synthesis of viral DNA, and 2) late mRNA, transcribed from progeny DNA, usually translates into viral structural proteins. Ex. Herpesviridae, Adenoviridae, and Papovaviridae. Class II _ ssDNA Single stranded negative DNA gene, make a single stranded positive copy, duplicate it and afterwards we have synthesis. Generally, the eukaryotic infective virus replicates mostly in the nucleus, through the rolling circle mechanism, forming a double-stranded DNA intermediate in the process. So, it makes a single stranded positive copy, duplicate it and afterwards we have synthesis. Generally, the Rolling Cycle is in a 3’-5’ end direction. In this category there are different types of viruses, the most important one is the Parvoviridae, which infects vertebrates. 16 Class III _ dsRNA In this class we have two major families: Reoviridae and Birnaviridae. Although, this class is not very well studied because we do not have very severe diseases caused by viruses of this class. As with most RNA viruses, this class replication occurs in the “Core capsid”, found in the cytoplasm, and does not have to use polymerases. Replication is monocistronic, we have an mRNA synthetizing a big poli- protein which, after being cleaved by viral proteases, makes functional structural proteins. (Replication includes individual, segmented genomes, meaning that each of the genes codes for only one protein, less complex translation mechanism). Class IV _ (+) ssRNA It is the positive-sense RNA viruses. All RNA defined as “positive-sense” can directly access to host ribosomes to immediately form proteins. It is like a real mRNA. (Works like a Rna, we have RNA replicases making the typical polyproteins which are then cleaved). One very important protein codified by the mRNA is RNA replicase, that makes negative strand that can be used as template to make more mRNA copies. Class V _ (-) ssRNA Includes single-stranded RNA virus with a negative-sense. They cannot be directly accessed by host ribosomes to immediately form proteins. Instead, they must be transcribed by viral polymerases (RNA- dependent RNA polymerase)- in fact we never find this type of polymerase in human cells, but it is already active in the viral particle - into a “readable” form, which is the positive-sense reciprocal. These viruses can also be divided into two groups: - Viruses containing nonsegmented genomes (ex. Paramyxoviridae, Filoviridae and the Rhabdoviridae) - Viruses with segmented genomes (ex. Orthomyxoviridae) 17 Transcribed by Viral RNA-dependent RNA polymerases, negative strand must become positive to start a real cycle. Orthomyxoviridae, (flu virus is a class 5), possess a segmented genome, every segment is a gene encoding for a single protein or part of it, which are later assembled. Class VI _ (*) ssRNA RT virus Starting from this class, we have a different strategy of replication. This class includes single stranded RNA positive sense, which, differently from the class IV, need a reverse transcriptase to make a cDNA that integrates in the nucleus of the host cell. In fact: - They replicate through a DNA intermediate - Use a reverse transcriptase to convert the positive-sense RNA into DNA (cDNA called also Provirus DNA). Instead of using the RNA for templates, they use DNA to create the templates, which is spliced into the host genome using integrase. Replication can then commence with the help of the host cell’s polymerases. - A well- studied family of this class of viruses includes the retroviruses (like HIV). Belongs to retroviruses, we start from a positive Rna, through retrotrascription we obtain a cDNA. Then the provirus integrates this segment in the host cell genome and starts the cycle. Class VII _ dsDNA RT virus This class includes double stranded DNA with two specific peculiarities: a reverse transcriptase and the genome is partially double stranded, meaning that it is incomplete. - This class replicates through a ssRNA intermediate, which is important for the pathogenic mechanism of hepatitis B, stimulating the formation of the interferon, which is important for hepatitis B infection. (Hepatitis B is an example.) - The Double-stranded, gapped genome subsequently filled in to form a covalently closed circle (cccDNA) that serves as a template for production of viral mRNAs and a sub-genomic RNA. - The pregenome RNA serves as template for the viral reverse transcriptase for production of the DNA genome. In fact, the RNA intermediate makes the cccDNA that forms the genome. 18 It must complete the segment first, then makes mRNA as an intermediate. The mRNA is later transformed into a negative DNA, reverse transcriptase makes a cccDna (covalently closed circle) so that the replicative cycle starts, transcription follows. NB. Generally, HIV integrates the genome, the Hepatitis B stays stable in the cells with the cccDNA (stable form inside the hepatocytes). Virus replication Cycle: ASSEMBLY Once all viral nucleic acid, enzymes and capsid proteins have been completely synthesized, assembly of components into complete virions begins. So, once all the different viral constituents are made, the new virus has to be released. RELEASE Assembled viruses can leave the host cell in 2 different ways: Budding or, Lysis. Budding: exocytosis of the new particle. It is typical of viruses with an envelope. In fact, the nucleocapsid binds the membrane which pinches off and sheds the virus gradually. (we know from last lecture that generally on the top of cell membrane we can find the proteins used for the linkage to the new cells). The cell is not immediately destroyed, these types of cells generally die by apoptosis mechanisms. Lysis: the cell dies, there is the complete lysis and the release of virus particles. It is typical of naked and complex viruses (no envelope), but some enveloped virus are also able to leave using this mechanism. The Virion is a fully formed extra cellular virus particle that is virulent (able to establish an infection in a host). We have a very high number of viruses released from the cell (depending on the life cycle, stability etc.), sometimes 3,000- 4,000 are released (pox virus) or in some cases they are more than 100,000 as in poliovirus. Release is a very important step. Some viruses can have a process of maturation and release enveloped viruses before the actual acquisition of infectivity (like the HIV virus). The viral proteins cut the glycoproteins present in the envelope of the virus, and after this type of cut we have the maturation, and the budding virion is released ad a free infectious virion with envelope. (On the net it says that enveloped viruses mature by budding at cellular membranes, which are then modified by envelope proteins). 19  Date: 13/02/2020 Lecture’s subject: Virus assembly and release, Professor: Santangelo pathogenesis, infection patterns, diagnosis Subject: Microbiology-Virology Sbobinator 1:Ankush Santosh Course: Biopathology I – First intermediate Sbobinator 2: Lucia Giacomobono This class we will talk about the pathogenesis of viral infection. Last week, we discussed Productive Infection cycle AKA the lytic cycle of the virus. We have 6 different steps – Adsorption, Penetration, Uncoating, Synthesis, Assembly and Release. (The text in the diagram gives a brief description of the steps). Only the interaction of the virus with the cell activates its pathogenic mechanisms. Generally, the virus doesn’t produce exoenzymes or toxins, doesn’t have a real secretion of different pathogenic proteins or any pathogenic mechanism. Only the interaction of the virus with a cell allows it to start pathogenesis. Sometimes instead of the productive/lyric cycle infection we can have a Non- Productive infection. The consequences of non-productive viral infections in animal cells can be divided into two types: Figure 1:Lytic Cycle 1)Active or acute infections 2)Persistent infections: release of virions form host cells without resulting in cell lysis and the infected cell remains alive and continues to produce new virus particles. Persistent infections can be further divided into two types: 2.1) Latent infections: The most common example is the herpes virus. There virus remains in the cells but there is no production of new virus particles. 2.2) Chronic infections: HIV virus is a typical example of chronic infection. There is a small amount of the virus being persistently produced with little or no damage to the target tissue. Non- productive infections are also capable of oncogenic transformation and cell fusion. Oncogenic transformation is the conversion of normal cells into tumour cells. Cell fusion refers to the fusion of two or more infected cells into one cell with multiple nuclei. One of the viruses which can give us all these types of infections is the EBV (Epstein-Barrett virus), and herpes virus. It can cause acute infection, then goes into latent phase/state (like all the herpes viruses) and can reactivate in determinate events and cause different types of diseases. It can also employ a direct oncogenic mechanism and cause different types of tumours. The picture recaps what is described so far. We begin, on the leftmost part, with a cell infected by a virus. We can have the following outcomes: Lysis of the cell with a productive infection and the production of new viral particles Figure 2: Outcomes of Viral Infection Oncogenic transformation into tumour cells  Chronic infection i.e., slow release of virus without cell death. Latent infection i.e., presence of virus but no replication. This latent infection may revert into a lytic infection which involves virus production and cell lysis. Cell fusion with multiple nucleated cells. Keep in mind that the persistent infections, both latent and chronic, are capable undergoing oncogenic transformation at some point. Cells infected with HPV, Papillomavirus, Hepatitis B, Hepatitis C are particularly susceptible to oncogenic transformation. Now we discuss the latent state of the virus life cycle. [She reads the slide]. Generally, the provirus is the complete genome of the virus integrated in the human/host cell genome. When the provirus eventually gets reactivated, there is the production of new viral particles and cell lysis. One of the viruses which have this kind of provirus infective mechanism is also HIV virus which can do both. Generally, it is more typical for EBV virus because for HIV there is directly 2 different types of cells which can be infected. In the figure, we can see the lytic pathway, which leads to cell lysis, and lysogenic pathway, which leads to the integration of the virus DNA inside the host DNA. After virus Figure 3:Latent State reactivation in the lysogenic pathway, it may enter the lytic pathway. [She reads the slide]. The susceptibility of the cells is dependent on the receptor for the virus. For a productive viral infection, we need a susceptible and permissive cell so that both viral attachment and replication can occur. They are the only cells which can take up a virus particle and replicate it and give us a new population of virus particles. They are the only cells that if infected can infect new cells and go on with the complete viral replication. Figure 4:Common Definitions of Infected Cells The latent period is very important during the viral infection. It is the time period from the beginning of infection until progeny virions are found outside the cells. The eclipse phase is another important concept. This phase is the period which goes from the interaction with the cell, and the entrance in the cell of the virus, and production of the first new virus particles. During the eclipse phase, the virus is absorbed and completely destroyed i.e., it loses its identity, and no real viral particle is present. The infectious virus only reappears at the end of the eclipse phase. A lot of viral infections Figure 5: One-Step Growth Experiment do not cause a real disease, there are some infections which cannot be cured at the beginning since the real disease appears after a lot of time (e.g. HIV infection - in normal conditions, with no therapy, 10 years are needed for the development of the real AIDS manifestation of the disease). When viruses destroy cells and increase in number inside the body, they cause disease. *For the next few sections, the professor only reads off the slide* Only the interaction of the virus with the cell activates its pathogenic mechanisms. Generally, the virus doesn’t produce exoenzymes or toxins, doesn’t have a real secretion of different pathogenic proteins or any pathogenic mechanism. Only the interaction of the virus with a cell allows it to start pathogenesis. [She reads the slide]. This is why have infection without a disease, i.e., an asymptomatic infection, most of the time. Viral pathogenesis contains six stages: 1. Transmission and entry of the virus into the host 2. Spread in the different sites of the body 3. Tropism, the interaction of the anti-receptor of the virus with the cell receptor Figure 6:Viral Pathogenesis 4. Virulence, depending on the different type of virus and on the conditions of the body 5. Patterns of viral infection and disease (acute, persistent, chronic one,...) 6. Host factors and defences. 1. TRANSMISSION AND ENTRY There are different routes of transmission, which can be divided in: Horizontal transmission: man-to-man (common route), respiratory, gastrointestinal, genitourinary, sexual routes. Vertical transmission: from mother to child, can occur both in uterus or during delivery (in birth channel) and through breast-feeding as well * Zoonotic transmission: animal-to-human transmission which can be direct or with different intermediate hosts (bites of animals - Rabies - and insects - dengue, yellow fever, West Nile etc.) *Placenta is a really good filter of everything especially in the first half of the pregnancy, then it becomes older, and something can pass through more easily. It allows passage of IgGs (IgMs and IgAs are produced by the fetus since they can’t pass through). Maternal milk instead contains everything we have in the mother’s Figure 7: Route of Entry and Examples body (drugs, different microorganisms and so on, as well as antibodies important for the newborn.  The table describes the routes of entry for different viruses. SARS-CoV 2 is transported by aerosol droplet inhalation. These respiratory viruses may cause respiratory diseases but sometimes they may only just be a route of transmission and cause other diseases. For example, Coronavirus, Influenza, Adenovirus etc directly affect the respiratory system but the Varicella Zoster virus can be transferred airborne and then affects the skin. [She then reads the all the route of entry plus a few examples from the table] Another important aspect of virus transmission is the incubation period. The incubation period is the time between exposure to the organism and appearance of the first symptoms of the disease. The virus generally multiplies at the site of entry in order to establish an infection in the host and only after, it can cause disease in the same site, or it can spread to the other organs. It can generally range from a few days (like influenza virus or other respiratory viruses) to a year. The table shows the incubation period within various viruses. The professor specifically mentions these ones – Influenza virus (2 days), HIV without antiviral therapy (years), Hepatitis B (30-160 days), Hepatitis C(15-150 days), papillomavirus(up to 10 years) Incubation period depends on the different type of Virus and on the different family of the virus. 2. SPREAD IN THE HOST After the entrance and the incubation period we have the spread in the host. We can have: Localized infection: The virus spreads mainly by infecting Figure 8:Incubation Periods and Examples adjacent or neighbouring cells, causing an acute infection. Most of the viruses in this case are the respiratory ones or gastrointestinal ones or skin infections. E.g., Influenza virus enters the host, replicates inside the respiratory epithelium cells, and generates an acute disease, after circa 1-2 week maximum all the disease ceases. When we have a new episode, everything starts again as the first infection (only if the immunization against that same virus is not life. Long). For a lot of these viruses, we have the formation of antibodies, and the immune response remains long-life. For other ones, depending on the family, immunity is not long lasting. Systemic disease: The virus spreads from the site of entry to the target tissue where it causes cell injury after multiplication. The most common routes to spread are - the Hematogenous route (by blood - in the bloodstream; example incl. HIV, Hep C etc.), the Neural route (examples incl. Herpes virus) and the Lymphatic route. 3. TROPISMTropism is the capability of the virus to infect a discrete population of cells within an organ. It is linked to the presence of a certain receptor on cell membrane. It is most often determined by the specific interaction between viral surface proteins and cell receptors. Different viruses may use the same receptor on host cells (e.g., CD4 is commonly used by HIV and HHV-6) and some viruses such as HIV can use a different receptor and co- receptor to enter different kinds of cells and make a productive viral infection. Involved receptors have a real function for the cells, the virus links to them and interacts with them to enter the cell. Table describes the different receptors used by different viruses. She specifically mentions the following – Adenovirus, Cytomegalovirus, Coronavirus (SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) and its mechanism is a bit different), EBV, HIV, Influenza virus. Figure 9:Virus Receptors and Examples 4. VIRULANCE AND PATHOGENICITY Pathogenicity is defined as the ability of a virus to cause disease in an infected host. Virulence is the relative ability of a virus to cause disease. Virulence can be measured as the degree of pathogenicity between closely related viruses to cause disease. Cytopathogenicity is the ability of a virus to cause degenerative changes in cells or cell death; Virulence and cytopathogenicity depend on the nature of viruses and the characteristics of cells (permissive and nonpermissive cells). The strict interactions between viruses and cells determine the diseases and symptoms. The more the ability to replicate and destroy cells the more the damage that a virus can cause inside the host. Cytopathogenicity is the ability of the virus to transform the cell, to destroy it or to cause deformation of the different cell components. When all these components are summated together the disease can go (flare) up and manifest the different specific symptoms. 5. PATTERNS OF VIRAL INFECTIONS AND DISEASE Infections are more common than diseases, a lot of times there is the infection without real symptoms or disease. Infection involves multiplication of the virus in the host, while the disease represents the clinical manifestation. The severity of the disease depends on the role of both viral and host factors in influencing viral infection and disease progression. Based on patterns and levels of detectable infectious virus in the host and the role of immune response in clearing the virus, viral infections can be divided into five categories:  Acute infection followed by viral clearance by the immune response (e.g., Influenza virus, Parainfluenza, Rhinovirus, Hepatitis A,...): Immune responses go up and can remain for life or be lost after different amounts of time. When there is a new infection, everything starts again. It is typical of respiratory viruses, in which there is an acute infection and an acute disease in 1-2 weeks, then the resolution. When there is contact with another virus or the same virus (in some cases) the cycle repeats again. For example, with the influenza virus, there is initially an acute respiratory infection. Eventually, this virus is cleared and resolved but once we come in contact with a new strain of influenza, the same symptoms show up again. Usually, but not always, with these types of infections, we have a protective long term immune response. We will inevitably need a vaccination to receive a high level of protection against the virus. For influenza, we perform the vaccination every year so that we have a very high percentage of people that have the antibodies against influenza. However, for Covid-19, we have not reached this point yet because the people are still with their initial inoculation, and they have antibodies which last no more than 5 months. Soon, after people complete their second inoculation, they will have a higher number of antibodies that offers better protection. Figure 10: 5 Categories of Viral Infections Acute infection followed by latent infection and periodic reactivation (typical of herpes virus and many other virus families): After the first contact these viruses remain inside the body in a different site for the lifetime. They remain inside the body life-long with some reactivation or no reactivation. Acute infection followed by a chronic infection (in Hepatitis B and C) : If there is no specific therapy, there is the continuous production of virus along with the expected signs and symptoms. Acute followed by persistent infection and virus overproduction (e.g., HIV): There is latent clinical phase of the disease but no latent viral phase of replication because without anti- retroviral therapy, the virus will keep replicating. Generally, the infection to the manifestation of the disease may take up to 10-20 years. At this moment, very high anti-retroviral therapy in seropositive conditions can help slowing down the symptom’s appearance (30-50 years without any symptom of AIDS).  There is an infection at the point 0 and AIDS can occur after 10 years, as in the natural history of the infection. Slow Chronic infections (can occur with different kinds of viruses, like some prions, polioviruses like the JC or PK): There is an infection early on in life, but any kind of manifestation or disease occurs much later in life. This entire process is incredibly slow. [She reads the slide].Generally, both DNA viruses and RNA virus can give eventually viral transformation either with oncoprotein formation or with indirect mechanisms that destroy the cells. Within this kind of virus, the most important regarding DNA ones are Papilloma viruses in which the oncoproteins produced completely change the cell cycle and in Human Herpes Virus like EBV and HHV-8 that is linked to Kaposi's sarcoma; occasionally also Poxvirus can be included in this category. EBV and HHV-8 interact with p53 to disrupt the normal cell cycle. While the RNA ones are Retrovirus as Human T-lymphotropic virus and Hepatitis B and C viruses (HIV is a retrovirus not Figure 11: Viral Transformation belonging to this class as it is not oncogenic). 6. HOST FACTORS AND HOST DEFENSE For immunocompetent patients, we have mild/no symptoms but for immunocompromised patients, the same virus may have very severe effects. Host factors such as immune status, genetic background, age, and nutrition play important roles in the outcome of viral infections. Age is incredibly important because we are most susceptible during the very early and very late stages of life. In the early period, the immune system is not completely formed and in the late period, the immune system does not function as well as before. Several innate immune responses and adaptive immune responses influence the outcome of viral infections. Some viruses cause severe diseases in infants; adults are more vulnerable to others. Host responses such as fever and inflammation have been suggested to have an important role in combating viral infections. Fever is really important in combating viruses because at high temperatures (39-40 degrees), the amount of virus being destroyed is much grater than the amount of virus being newly replicated. Other responses include the interferon production, interleukin production, enzyme production etc. Other factors that influence the host defence are socio-economic status, genetic background, age, nutrition, innate immune response, adaptive immune response. Regarding viruses the most important are IgGs that can interact and block the interaction of the receptor to the anti-receptor, this process is sometime exploited by vaccines that protect us from the virality of the virus. DIAGNOSTIC VIROLOGY Virologic diagnosis can be divided in direct and indirect, the latter generally is based on specific antibody detection (IgG, igM, sometimes igA) to see the status of the immune response. When we talk about seropositivity is not just referred to HIV, but it is when we have the antibodies against whatever type of viruses meaning that we have been in contact with that virus. Seropositivity can last for different amount of time depending on the viruses: herpes family give a long time immunity (all lifelong), the same for Chickenpox virus; while for Hepatitis A1 virus the immunity can be lost over time. This relays on the quality of the memory cells that depends on the type of virus and also on the different type of disease that it can cause. The direct one comprehends: Cell culture: we can look at the cytopathic effect directly when viruses grows within the cells and then we can isolate and identify them. To do this we look at the nucleic acid Fig.12 of the virus. Microscopy: electron microscopy Antigen detection: important for the identification after the cell culture or directly from the human sample. Nucleic acid detection: this method is the most used nowadays, with this method we can, for example, look at the specific production of a protein. She read the slide Fig.13 SEROLOGY (She read the definition on the slide) In serology aside from looking at the IgMs in primary infection, we can also look for seroconversion (when we have the appearance of IgMs and/or IgGs in the second sample of the same patient), this phenomenon is obviously detected later on. Usually for the immunospecific response the first antibodies during viral infection are igMs, after one month (2 to 4 weeks) they decrease and at the same time the igGs rise. The latter often show positivity for a long time. Generally when we talk about seropositivity is not just referred to HIV, but it Fig.14 is when we have the antibodies  against whatever type of viruses meaning that we have been in contact with that virus.Seropositivity can last for different amount of time depending on the viruses: herpes family give a long time immunity (all lifelong), the same for Chickenpox virus; while for Hepatitis A1 virus the immunity can be lost over time. This relays on the quality of the memory cells that depends on the type of virus and also on the different type of disease that it can cause. Sometimes for viruses they affect mostly the mucosa, we can find the appearance of IgAs, so when we suspect this kind of Figure 15 infection we can check the IgAs to detect a serious inflammation. TIMELINESS OF THE DIAGNOSIS It depends on the test chosen (depending on the test it could take up to 4 weeks): Culture: Needs to be set up during the acute stage of illness - may take days or several weeks for a diagnosis. It depends on the replication cycle of the virus. While the herpes virus can grow up in 1-2 days, the cytomegalovirus needs 7-10 days to have a real cytopathic effect. Antigen/Nucleic acid: present only during the acute stage of illness – usually quick methods for diagnosis and also to look at the different amount of the virus that can circulate. It is very important also to check what is going on during treatment. Serology: May not be present until after symptoms have resolved (generally 2-3 weeks depending on the incubation period). Is usually a quick method for virus detection. VIRUS ISOLATION Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures: 1. PRIMARY CELLS: come out directly from the tissue and generally after 2-3 passage the cell dies. 2. SEMI-CONTINUOUS CELLS: usually the diploid one, the more used are Human embryonic kidney and skin fibroblasts, it could be the 3. CONTINUOUS CELLS: are transforming cells that undergo to different passage directly in culture, generally infected by an oncogenic virus, so that we can make a lot of passages without loosing cells to senescence VIRUS TRANSPORT MEDIA It is important to have a good cell culture is the virus transportation medium that needs to stabilise the virus and to maintain the output of the virus to simulate the real infection with the interaction between receptors and anti-receptors. When the virus grows, we can see the cytopathic effect (when the virus infects the cell mono layer), a typical transformation of the cells that can be observed with the microscope and then, with the immunofluorescence trough specific antibodies, with this method we can recognise the type of virus. Another Fig.16 Effects can be the heamadsorption , in this case they acquire the ability to stick onto mammalian red blood cells. Figure 17 Figure 18 IDENTIFICATION The neutralisation, that is a specific antibody that prevents the cytopathic effect (CPE), with this procedure we can identify and prevent the infection in specific type of cells. Tough the most common procedure to eventually identify the specific virus is the immunofluorescence that can be direct or indirect. The direct approach utilises a specific antibody that is already linked to an antigen and to and indicator (look at fig.19). In the indirect approach (used when we want to identify the specific virus) we use a specific mouse antibody and in the second step we utilise an anti mouse antibody liked to an indicator. The most utilised one is the direct one. Figure 19 PROBLEMS WITH CELL CULTURE Growth and detection of viruses using cell cultures present many difficulties: Long period for results: herpes viruses and influenza are the faster ones, but many virusesneed between 2 to 4 weeks to grow and show a cytopathic effect. The problem with this is the time of growth, so in order to make faster diagnosis we utilise the molecular technique. Poor sensitivity: this depends for example on the quantity of the sample, but on the other side  specificity is very high. Bacterial contamination: especially for samples coming from locations with high bacterialpresence (e.g. genital tract). Presence of toxic substances: sometimes it is impossible to start a cell culture from stools orblood because there are substances toxic for the cell line. Many viruses do not grow in cell culture: like HBV and HCV which can grow only in animalmodel, so sometimes we don’t have the appropriate mono-layer to grow this kind of cultures. RAPID CULTURE TECHNIQUES They can be used only for some type of viruses which start to produce detectable antigens 2/4 days after the inoculation. The antigens will be detected using immunofluorescence. This technique is used mainlyfor the CMV with DEAFF detection (this is an obsolete technique nowadays), which is a virus that usually takes more than one week to grow completely in cell culture. In the image we can see the viruses that can be isolated with the cell culture. Fig.20 ELECTRON MICROSCOPY (completely obsolete for clinical virology) This technique is not very efficient, because we need many virus particles in the sample (106) and sincewe can recognize only the morphology of the virus (which is the same for a whole virus family) we cannot exactly identify the species contained in a sample (low specificity). In the image below (fig.21) you can see two micrographs, the first one of an adenovirus and the second of a rotavirus. They are viruses without envelope, which present only the capsid. The problems with electron microscopy are: Expensive equipment, Expensive maintenance, Requires experienced operator, Sensitivity and specificity are low Fig.21 above; fig.22 below SEROLOGY This is an indirect way of virus identification, because we are looking at the immune response of the host and not at the presence of the virus. CRITERIA FOR DIAGNOSING PRIMARY INFECTION 4-fold or more increase in titre of virus-specific IgGs or total antibodies between acute and convalescent sera. Presence of both IgMs and IgGs, which in many cases indicates a primary infection. Presence of seroconversion, that means that after having a sample negative for virus-specific antibodies or having only IgMs against that pathogen, we can find a second sample positive forvirus-specific IgGs. When seroconversion is present, we are surely in front of a primary infection. A single high titre of IgGs or total antibody, but it is unreliable if an avidity test is not performed. CRITERIA FOR DIAGNOSING REINFECTION An increase in titre of IgGs or total antibodies between acute and convalescent sera. Absence or slight increase in IgMs. Note that for the reinfection we only need an increase in antibodies' titre, without a quantification. It islike this because sometimes the titre can vary in a very narrow range due to the presence or the slight increase of IgMs in serum. ELISA for HIV antigen and antibody Generally, the most common serological test used is the ELISA (immuno-enzymatic system) to detect HIV infection. ELISA can be used to make diagnosis both in a direct (detection of viral antigen) and indirect (detection of virus-specific antibodies). For HIV testing with ELISA we have the antibody capture and the Antigen capture, the main difference is that in the antigen capture we have blocked the antigen in the solid support and then we can insert the clinical sample. If the viral antigen is present it is liked to the antibody and with the addition of an indicator we can make the diagnosis.We usually have a first screening test that has a very high sensitivity and a good specificity (antibody ELISA), then the diagnosis must be confirmed through Western Blot (that has good sensitivity and a high specificity). It is a test used to confirm the first result, where we use different antibodies specific for viral proteins and glycoproteins to identify them in the sample (figure on the right, the black bands arethe one where antibodies are bound; all the viral protein are sorted out looking at their molecular weight). In the most modern HIV tests we are able to detect at the same time virus-specific antibodies and the p24 antigen, which is present when the viral cycle is going on. In conclusion to make the right diagnosis, we need to Fig.23 have at least the recognition of two different antibodies. ( she said that when we will do the HIV we will go in depth with this point)  UTILITY OF SEROLOGY First of all we need to know how useful the serological results are, for viruses such as rubella and hepatitis A where to onset of the symptoms coincide with the development of the antibodies, the serological test is really important. In other viruses the serological test is important to understand if we have a primary or secondary infection (that can be either a reinfection or a reactivation of the virus). PROBLEMS WITH SEROLOGY (she read the slide, so here’s the slide) Fig.24 RAPID DIAGNOSIS BASED ON DETECTION OF VIRAL ANTIGENS This molecular technique is very important for immunocompromised patients that don’t have a strong hum oral response, therefore to be sure of the diagnosis that we made we need a more precise test. This is test is carried out on samples coming from specific places for different viruses. It is especially useful for respiratory and gastrointestinalviruses. In all these cases we try to detect a specific viral antigen through immunofluorescence or any immuno technique to individuate the antigen we are looking for. In the images below we can see a sample of immunofluorescence made for a herpes virus infection (on the left and for CMV (on the right) Fig.25. Above; Fig.26 below ADVANTAGES Results are quickly available (within few hours) POTENTIAL PROBLEMS Reduced sensitivity compared to cell culture, that can be as low as 20%. Specificity is also poor. Requires good specimens  The procedures involved are often tedious and time-consuming and thus expensive in terms oflaboratory time. Another important thing to take into consideration is to take the right sample to make the right diagnosis. For example if we have the suspect of a respiratory infection the first test to perform is a nasopharyngeal swab. SPECIMENS FOR ROUTINE TEST In the table we can see what tests are useful in function of the virus we are searching for. Then she proceeded to read the table. Fig.27 MOLECULAR METHODS Recently we have started to use also molecular methods to have a diagnosis of viral infections. Molecular methods are rapid and have a high sensitivity and specificity. These methods are based on the detection of viral genome, they are the future direction of viral diagnosis. Nowadays, their use is increasing in clinical practice, but their role is still small compared to conventional methods. Their use will increase in the near future. We have classical and new-generation molecular techniques. CLASSICAL MOLECULAR TECHNIQUES They generally look at viral genome (DNA or RNA) and sometimes also to viral proteins production and expression. The most common are the Dot-blot and in-situ hybridisation, the use of this techniques depends on the specific DNA/RNA probs, if we want to look for the formation of a specific protein we also utilize the mRNA. However since they are very expensive and time consuming they never found widespread acceptance. POLYMERASE CHAIN REACTION (PCR) In the image below she read what are the advantages and disadvantages of the PCR. For viruses it is a more complicated procedure compared to the one for bacteria. In fact, for bacteria wehave common primers that can be used with many bacterial species and strands, while for viruses we need specific primers for every type or viral family. This technique is very sensitive, easy to set up and it has a fast turnaround time. Its main disadvantage is the liability to contamination, in fact PCR requires a lot of care during its set-up. Fig.28  SCHEMATIC OF PCR For the PCR (reverse transcriptase PCR) you have at first a target RNA, which is then transcribed in a cDNA which will be amplified in the standard way. Fig.29 OTHER MOLECULAR TECHNIQUES Branched DNA is a technique in which the signal is amplified in a linear way instead of anexponential one. There is no amplification of the genome. The NASBA uses very small oligonucleotides to link in a specific way the different targets andamplify the signal. PCR is still the technique with the higher sensitivity. In this image we can see the different molecular methods with they’re qualities, pros and cons. Fig.30 REAL TIME PCR (most utilised) In this case the sample is mixed also with DNA probes, which when hybridized with the target DNA will give a positive signal. This technique enables us to see and quantify in real time the presence of thevirus in the sample. To do this we need the standard curve which tells us the starting quantity of viruses in the sample looking at the number cycles which were necessary to the real time PCR to find a positive result. There is a difference tough between liquid and solid samples, in Fig.31 fact for the liquid one we can make the quantification directly in the international metric system (and for the solid one she didn’t say anything). This is important to assess the severity of viremia or to understand if an antiviral therapy is working.  DNA SEQUENCING It is important to make identification of mutant strands which have developed drug resistance (especially for HIV, HCV, HPV and corona viruses ). This test is really important, for example, to establish the antiretroviral therapy for HIV patients. GENE CHIP (fig..) It is made up of probes which are linked to a solid support, after that we can insert labelled DNA copies from the sample and when they hybridize with the probes, we can identify the virus itself or its mutations. Fig.32 ALLPLEX PANEL ASSAY (very utilized nowadays) With 4 different panels we can look at different types of viruses and also to some bacteria directly in the same sample. In the automated process you can go in less than 6 hours directly from the specimen, to the automated extraction and PCR set-up, and after that quantify the virus presence with real time PCR. Fig.33 ALLPLEX RESPIRATORY FULL PANEL ASSAY With this kind of these. We have only three passages since the specimen is inserted in an automated machine and we have a real time PCR of a lot of samples simultaneously. Fig.34  DATE: sbob 3 - 10/01/2022 COURSE: Biopathology 1 PROFESSOR: Santangelo Rosaria MODULE: Medical Microbiology (Virology) TOPIC: Retrovirues - HIV BOOK REFERENCE: chapter SBOBINATORS: Manzato - Dargenio REVIEWER: mais oui Retroviruses Retroviridae is a big family of viruses, among which the most important in human pathogenesis is HIV. The Retroviridae is a family of enveloped (+) sense ssRNA viruses that have been intensely studied because of their association with cancer (such as leukemia), and AIDS (acquired immunodeficiency syndrome). Oncoviruses are completely different from HIV, since this last causes AIDS. In this case, cancers and other diseases stem from the immunodepression. Historical background The first association of viruses with cancer was in early 1900’s with the discovery by Ellerman and Bang that leukemia could be transmitted from one chicken to another by injecting leukemia cell extracts. In 1911 Peyton Rous showed that a bacterial free filtrate (meaning it has virus inside) from solid tumors of chickens could cause an identical cancer in chickens inoculated with the filtrate. The virus causing the leukemia was subsequently shown to be avian leukosis virus and the virus causing tumors was called Rous sarcoma virus. Although the discoveries by Ellerman, Bang and Rous were not well accepted at the time, 60 years later these viruses were designated retroviruses and Rous won the Nobel Prize for his work in 1963 at the age of 83. In early 1970, Baltimore and Temin independently identified the unusual enzyme, reverse transcriptase and won the Nobel Prize in 1975 for their work. It subverted completely the classical mechanisms of transcription and translation. Generally we have the formation of Protein starting from DNA passing through RNA intermediates. In this case starting from an RNA, we form a cDNA intermediate and from here the classical cycle. cDNA is then integrated in human genome and will proceed to transcription and translation. Their discovery shattered the central dogma of molecular biology which stated the flow of genetic information was from DNA to RNA (we can go from RNA to DNA through reverse transcriptase, thus DNA can integrate in other cells’ genome). In 1989, Bishop and Varmus won the Nobel Prize for elucidating that retroviral oncogenes are derived from cellular genes and brought us closer to understanding cancer. The first case of AIDS was reported during 1981 in the USA by Antony Fauci, an immunologist. The First time, Gallo discovered in 1981 the retrovirus (HTLV-Human T Cell Leukemia Virus III). Only later, by Montaigner and Barre-Sinoussi in 1983, HIV was isolated. 1 Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. This large and diverse family includes members that are oncogenic. It is associated with a variety of immune system disorders and causes degenerative and neurological syndromes (either by direct interaction or by indirect mechanisms involving different organ tropisms). These viruses are species-specific (human to human, animal to animal). Some members are pretty much oncogenic, other act only at the level of immune system. Classification: - Retro (Latin) – backwards - Onco (Greek, oncos) – tumor - Spuma (Latin) - foam - Lenti (Latin, lentus) – slow; the name is due to the time between infection and disease, not because of a slow replication cycle. In HIV 10-20 years can pass. With ART (antiretroviral therapy), it takes more time for the disease to appear. Fig.1 Retrovirus Genus classification Among retroviridae, the most important ones for humans are Deltaviridae and Lentiviridae At Electron microscopy we can observe a core with a capsid, usually with icosahedral symmetry, and an outer envelope. Fig.2 Lentivirus (HIV) at TEM There are two major groups of retroviruses that infect humans: - Oncoretroviruses (onco-, “related to a tumor”) - HTLV 1, 2, 3, 4. They cause tumors in many animals (leukemias, lymphomas and sarcomas) and were discovered in 1970. HTLV-I and HTLV-II are associated with human leukemias/lymphomas strictly; Oncoviruses are usually not cytolytic, but produce or activate oncoprotein that can alter the cell cycle. - Lentiviruses (lenti-, “slow”) - HIV‐1, HIV-2. Among these we can also see different subgroups and quasi-species. Lentiviruses can apparently persist in infected hosts for long periods of time in a clinically latent state (“clinically” because even though there is no disease, there are a lot of different of particles replicating in the body; with therapy replication can be stopped). Over time, the virus becomes highly cytopathic and kills infected T-cells and also uninfected T- cells, causing impairment of the host immune defenses, followed by AIDS and opportunistic infections. There are two types, HIV-1 and HIV-2: 2  § HIV-1 is the more pathogenic one and the major cause of AIDS worldwide, § HIV 2 is less pathogenic and mainly present in less developed countries. Other groups of retroviruses infect animals. The specificity for certain animals derives from the difference in receptors present between animals and humans Endogenous retrovirus sequences are found throughout the human genome. HIV – Human immunodeficiency virus In 2008, two French virologists, Françoise Barré-Sinoussi and Luc Montagnier, shared the Nobel Prize in Medicine for their work on the discovery of HIV, the virus that causes AIDS. “The most important disease resulting from a human retrovirus infection is called acquired immunodeficiency syndrome (AIDS), which is caused by a lentivirus known as Human Immunodeficiency Virus (HIV)” HIV and all other retroviruses are remarkably similar in their basic composition and structure, in fact the virion contains: - Two identical single stranded positive-sense RNA molecules (we can say diploid genome even if it is not a eukaryotic cell). Even though it is a positive strain RNA it cannot directly act on translation process since it is not and mRNA. We need a reverse transcriptase to form first a cDNA in order to produce a real mRNA. - It carries enzymes in the virion that are essential for replication. Indeed, inside the virion we have directly the reverse transcriptase and integrase which already functional, while there is also a protease that is inactivated but will become active later on in the cycle. Many minerals and tRNA which are very important to start reverse transcription - it is an enveloped virus: on the envelope there are proteins that can bind to the receptor on the host cell. It is very important for the tropism of the virus and the different cell lineages it can infect based on the receptor types The virion size is about 100 nm in diameter and the RNA genome is coated with the nucleocapsid protein (NC). - The RNA–proteins complex are enclosed in a capsid (CA), also called p24, very important for diagnostic purposes since it is detectable when the virus is in active replication. This capsid is composed of multiple subunits in an icosahedral symmetry, which is covered by a membrane- associated matrix (MA), also called p17 protein. Fig.3 Structural features of HIV Indeed, in HIV, we have the capsid and the envelope, with the membrane in the middle Note: if it is a RNA virus we call this layer Matrix. If it is a DNA virus it is called tegument 3 - The envelope is acquired during budding from the host cell plasma membrane, but the surface proteins (SU), also called gp120 and the transmembrane protein (TM), also called gp41 glyco-proteins, found in the envelope, are virally encoded and maturated by the proteases action during viral cycle. Gp120 is the antireceptor for CD4 while gp41 is a fusion protein. Fig.4 Attachment modality of HIV to host cell Genome structure It is divided into fundamental and accessory genes: Fundamentals genes are: - Gag: encodes for the matrix, capsid and nucleocapsid -> structural proteins - Pol: encodes for reverse transcriptase, protease, integrase and RNAase H (different enzyme with respect to reverse transcriptase. This differs from Hepatitis B virus, where reverse transcriptase and RNAase H are combined) -> functional proteins - Env: surface glycoprotein and transmembrane glycoprotein -> envelope for adsorption and diffusion of the virus Each genes encodes a polyprotein which is subsequently processed by a viral protease (in most of the case) to yield individual functional viral proteins. LTR (long terminal repeats regions) are present at each terminus of the genome. They are about 600 nucleotides long and are important in integration of cDNA in the host genome. they can detect complementary regions in cells genome and integrate with it. Proteases are important for cleavage of new viral particles. Fig.5 HIV genome structure Regulatory and accessory genes There are many regulatory and accessory genes which requires mRNA splicing, and all apparently encode proteins that serve regulatory or accessory roles during the infection. 4 She then reads the whole table Fig.6 Accessory genes and associated proteins - functions Virus replication Life cycle: 1. Virus attaches to host receptors through the SU gp120 glycoprotein, resulting in a strict membrane-membrane linkage, while TM gp41 glycoprotein mediates fusion with cell membrane 2. ssRNA(+) genome is copied into a linear dsDNA molecule by the reverse transcriptase; 3. Nuclear entry of the viral dsDNA which is covalently and randomly integrated into the cell’s genome by the viral integrase (=provirus integration). In this step the LTRs are fundamental. 4. Transcription of provirus by Pol II (polymerase II) produces viral spliced and un-spliced RNAs; 5. Nuclear export of the incompletely spliced RNAs. 6. Assembly of the virion at the host cellular membrane and packaging of the viral RNA genome. 7. Budding through the plasma membrane and release of the virions (release of HIV particles can occur either by budding or by lysis). 5  Fig.6 HIV replication HIV entry HIV needs a receptor and a co-receptor: only when these 2 linkages are completed, the virus will enter the host cell. Gp120 binds to CD4, which is present on T-helper cell lineages and macrophage-monocyte lineage (MML). This binding will produce lytic events in T-cells and apoptotic events in the MML. In order to have a real binding in the cell we need a co-receptors. These are: - CCR5 mainly present in MML - CXCR4 in T-helper cells From here we can distinguish two classes of HIV: - R5 HIV-1 binds to CD4 and CCR5, - X4 HIV-1 interacts with CD4 and CXCR4. HIV-1 surface glycoprotein gp120 is divided in 3 domains: V1, V2, V3: - V1 and V2 attach to CD4 receptor of cells (lymphocytes and MML) - V3 interacts with the co-receptors, CCR5 or CXCR4. Fig.7 Adhesion mechanism on T-cell and MML From previous year sbob: “There is a group of persons called “non-responders”: they are 6  immunized against HIV since they have mutations in CCR5. This was discovered during trials for a new antiviral drug, Maraviroc, which is an antagonist of CCR5; blocking the linkage to co- receptors, HIV is not able to enter the cell, since the only link to CD4 is not enough.” Transmembrane gp41 protein mediates fusion of viral and cellular membranes. This doesn’t link any receptor or coreceptor, it is only a fusion membrane. The CD4+ T-lymphocytes express higher levels of CD4 and CXCR4 and somewhat lower levels of CCR5; however, the monocytes/macrophages express lower levels of CD4 and CXCR4 but higher levels of CCR5. Tropism is paramount to understand the pathogenic mechanism. - M-tropic are present in the first part of the infection (at transmission) and in latent phase. They respond first since it affects mainly the MML, which are spread in multiple body sites, while T cells are only in blood. Usually, it causes death by apoptosis. - T-tropic is present in late phase of disease and it is highly cytopathic (kills by lytic events) - Dual-tropic: present from the beginning to the end. Same virus can link both CCR5 and CXCR4 - Dual mixed: in the population of HIV virus, some can bind CCR5, other can bind CXCR4. When looking at HIV patient this is what we can observe: - At the beginning the infection will be in Monocytes and macrophages - Later, T-cells will be infected. Generally T-cells are destroyed and tend to decrease in number. If HIV can only infect this cells, the infection will stop and the disease will not progress. Conversely, if they infect macrophages, they act as reservoirs in different body sites, with the ability of continuously produce new viral particles. Fig.8 HIV tropism and disease progression 7  Viral postentry events Soon after entry, the virus partially uncoats. The reverse transcriptase copies the RNA to a dsDNA, which will then integrate into the host chromosome by the means of a DNA integrase, and will