User:Kinkreet/Virology

Introduction

There are more than 10³⁰ bacteriphage particle in the world's water supply. Every mililiter of seawater has more than a million virus particles.

There are currently ~10¹⁶ HIV.

The mass of a typical bacteriophage particle is ~10^-18 kg.

Viruses produce a huge number of progeny, and thus gives rise to a large number of mutants. Natural selection removes the less-adapted mutants leaving the well-adapted mutants to survive and further replicate. Because of the fast rate of replication, as well as the large number of progeny, viruses are very quick to evolve.

Classification of a virus depends on the nature of the genome (DNA or RNA), the symmetry and dimensions of the virion and capsid, the presence or absence of a envelop (lipid membrane), and genomics.

The Baltimore system is a classification of viruses based on the genome. There are 7 different classes according to this system: dsDNA, gapped dsDNA, ssDNA, dsRNA, ss(+)RNA, ss(-)RNA, and ss(+)RNA with DNA intermediate.

We carry viral genomes in the human genome, presented as retroviral genomes that were integrated into the DNA. This makes up ~8% of all our DNA. Thus, we have all the information in our genome to make a complete virus, but most are defective and have mutations in them, and so do not produce virus particles.

We are colonized by adenoviruses, coronaviruses and rhinoviruses. Bacterial flora found in the gut which are beneficial for humans, harbor their own viruses.

The endogenous retroviruses infects everyone. Anelloviruses are DNA viruses that seems to infect everyone and have unknown functions.

As the respiratory and gastrointestinal tracts form the interfaces with the outside environment, they are the most likely entry sites of viruses. Thus, these are also the areas where the immune system is most active.

Viruses can infect anything that is living, and can cross from one species to another species, a feature known as zoonotic infections.

Whales are common infected with small RNA viruses Caliciviridae family, which can cause diarrhea in whales and can infect other marine animals and humans, causing rashes, blisters and gastroenteritis. These viruses in turn replicate in the whale; infected whales secrete more than 10¹³ calciviruses per day.

Despite the abundance of viruses, because all organism evolved in parallel to viruses, they have evolved immune systems to fight the infection. When the immune system is down, such as in AIDS or patients under post-transplantation immunosuppresants, the viruses are no longer removed, and can cause lethal infections.

All viruses fundamentally consists of a genome inside a particle. The particle protects the genome as it spreads from one cell to another. The genome contain all the information to make another virus and ensures the infectious cycle is carried on.

All viruses are obligate molecular parasites, which requires a host to function. However, there are different dependency on the host; if a virus contains only a few genes, it relies on the host to produce much of its required gene products, compare this to a virus with many genes, able to produce much of itself from its own genome. Most viruses contain less than 15 genes, and some even only has 1 gene.

Regardless of whether the viral genome is DNA- or RNA-based, all viruses must synthesize mRNA and is translated by host ribosomes. No virus contains all the replication machinery and must rely on the host machinery one way or another.

A virus is a small, infectious, obligate intracellular parasite. A 'virion' is an infectious particle.

Classes of virus

DNA viruses

Replication of all DNA viral genomes cannot occur straight away, and requires the expression of at least one viral protein, often more. This means that transcription of the DNA genome, and the translation of the derivative mRNA, must occur prior to replication. And because transcription and translation must always rely on the host machinery at some stage, the replication of the DNA viral genome must, therefore, indirectly dependent on the host. And often, the host provides other proteins directly involved in DNA replication. Because of this, viruses do not replicate well in quiescent cells. To counter this, the virus can transcribes proteins which promotes cell cycle progression, such as host replication enzymes and cell cycle regulators.

DNA is synthesized semi-conservatively in a 5' → 3' direction, and requires a primer (RNA, DNA (hairpin structures) or proteins (covalently attached to the 5' end)) at a defined origin of replication. The origin of replication (Ori) usually contain A/T-rich DNA. This because A/T hydrogen bonding is weaker than C/G bonding, and thus is easier to open.

Apart from Poxviridae, which replicates its DNA in the cytoplasm, all other DNA viruses replicates their genomes in the nucleus. Replication occurs in replication centers, which consists of a the DNA template complexed with replication proteins. There are only a limited number of discrete sites, and is thought that the maximum number is 10. Nuclear domain 10s (ND10s) or PML bodies, or PODs, are scaffold that brings together polymerases, ligases, helicases, topoisomerases.

ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, is one of seven proteins encoded in the viral genome of HSV-1 that is required for HSV-1 DNA replication. It is able to anneal to ssDNA as well as melt small fragments of dsDNA; its role is to destabilize duplex DNA during initiation of replication. It differs from helicases because it is ATP- and Mg²⁺-independent. In cells infected with HSV-1, the DNA in those cells become colocalized with ICP8.

Small DNA viruses, such as Papillomaviridae, Polymaviridae and Parvoviridae do not encode an entire replication system; large DNA viruses such as 'Herpesviridae, Adenoviridae, Poxviridae, encode most of their replication systems. These proteins include origin binding protein (OBP), which recognizes specifically viral genes; helicase, primase, polymerase and accessory proteins (makes the polymerase more efficient). Exonucleases are often encoded to monitor the replication process as well as to remove the primers. Some virus also encode proteins involved in deoxynucleotide triphosphate metabolism; cells at rest have low levels of thymidine kinases, and thus do not synthesis TTP, the virus now provides the cell with this protein and use the nucleotides for its own use.

The DNA genome can be replicated into new viral progeny and escape the host cell to infect other cells (lytic infection), this usually results in a high copy number because the cell it has infected needs to be overwhelmed by the virus in order for the cell to become compromised and the virus can escape.

However, the virus can also integrate into the host genome and will divide when the host divides; or it might just remain in the cell as a plasmid-like structure, and is replicated with the chromosomal DNA because they have proteins attached that makes it appear to be a chromosomal DNA. This is termed latent infection.

dsDNA

Herpesviridae

Herpesviridae
Virus classification
Group:	Group I (dsDNA)
Order:	Herpesvirales
Family:	Herpesviridae
Genera
Subfamily Alphaherpesvirinae Iltovirus Mardivirus Simplexvirus Varicellovirus Unassigned Subfamily Betaherpesvirinae Cytomegalovirus Muromegalovirus Proboscivirus Roseolovirus Unassigned Subfamily Gammaherpesvirinae Lymphocryptovirus Macavirus Percavirus Rhadinovirus Unassigned Subfamily Unassigned Unassigned

Once infected with a herpesviruses, you are infected for life. There are 9 known herpesviruses - HSV-1, HSV-2, VZV, HCMV, EBV, HHV-6, HHV-7, HHV-8 and B virus. The B viruses is 100% lethal for humans. We are infected with ~3 of the 9 known herpesviruses - HSV-1, VZV, EBV.

Herpesvirus are 2000Å virions with over 80 genes, over half of which encode for proteins. Of these, 13 encodes for envelop proteins. Four genes encode for proteins that surrounds its DNA genome to make the icosahedral nucleocapsid. And 20 genes encode for an internal structure called tegument, involved in delivering proteins required in early infection.

On the capsid of Herpes Simplex Virus, there is a portal, or opening, one per virion, which is used for transport of the viral DNA genome out of the virion. The portal can be found on one of the 12 vertices of the capsid.

Herpes virus have a large, linear DNA genome, and also have inverted and terminal repeats. They have unique origins of replication; Herpes simplex virus have three origins of replication, two of which are in these repeats (they are the same), and one in the long (L) region. These origins have stem loops which ensure basepairing.

Adenoviridae

The adenovirus uses a protein molecule called terminal protein to initiate replication.

Polyomaviridae

Kinkreet/Virology
Micrograph showing apolyomavirus infected cell - large (blue) cell below-center-left. Urine cytology specimen.
Virus classification
Group:	Group I (dsDNA)
Family:	Polyomaviridae
Genus:	Avipolyomavirus Orthopolyomavirus Wukipolyomavirus
Species
Avian polyomavirus Athymic rat polyomavirus B-lymphotropic polyomavirus Baboon polyomavirus 1 (SA12) Baboon polyomavirus 2 Bat polyomavirus 1 BK polyomavirus Bornean orang-utan polyomavirus Bovine polyomavirus California sea lion polyomavirus Canary polyomavirus Chimpanzee polyomavirus Crow polyomavirus Cynomolgus polyomavirus Finch polyoma virus Goose hemorrhagic polyomavirus Gorilla gorilla gorilla polyomavirus 1 Hamster polyomavirus Human Polyomavirus 6 Human Polyomavirus 7 Human Polyomavirus 9 Human Polyomavirus 10 JC polyomavirus KI polyomavirus Mastomys polyomavirus Merkel cell polyomavirus Murine pneumotropic virus Murine polyomavirus MW polyomavirus MX polyomavirus Rabbit kidney vacuolating virus Pan troglodytes verus polyomavirus 1a Pan troglodytes verus polyomavirus 2c Simian virus 40 Sumatran orang-utan polyomavirus Squirrel monkey polyomavirus Trichodysplasia spinulosa-associated polyomavirus WU polyomavirus

Polyomavirus have a circular, supercoiled dsDNA genome. Because of the supercoiling, there is torsional energy which causes the genome to twist.

Mimiviridae

Mimivirus
Virus classification
Group:	Group I (dsDNA)
(unranked):	Nucleocytoplasmic large DNA viruses
Family:	Mimiviridae
Genus:	Mimivirus
Species
Acanthamoeba polyphaga mimivirus

The mimivirus is one of the largest known virus, it was isolated in a water tower in France. Its natural host is the ameoebae. It consists of a 500nm particle with 125nm fibres extending from it, giving an overall diameter of ~750nm. It has a double-stranded DNA genome of 1181404 base pairs which encodes for 1262 open reading frames. It encodes many components of the protein synthesis pathway, including amino-acyl tRNA synthetases, peptide release factor 1, translation elongation factor EF-TU, translation initiation factor 1, 6 tRNAs, Type I and II topoisomerases, DNA repair complexes, and many polysaccharide synethesis enzymes.

ssDNA

Parvoviridae

Parvoviruses
Virus classification
Group:	Group II (ssDNA)
Family:	Parvoviridae
Subfamilies
Densovirinae Parvovirinae

Parvovirus

Parvovirus
Virus classification
Group:	Group II (ssDNA)
Family:	Parvoviridae
Genus:	Parvovirus
Species
Bovine parvovirus Canine parvovirus Chicken parvovirus Feline panleukopenia virus Feline parvovirus Goose parvovirus Hamster parvovirus HB virus H-1 virus Human PARV 4 Human PARV 5 Kilham rat virus Lapine parvovirus LUIII virus Mink enteritis virus Minute virus of mice Murine parvovirus 1 Porcine parvovirus Porcine hokovirus Raccoon parvovirus Rat minute virus 1a Rat minute virus 1b Rat minute virus 1c RT parvovirus Tumor virus X

A parvovirus is a single-stranded DNA virus, but it can come as either (+) stranded or (-) stranded, but only one type is associated with each virion. This is problem for replication. This is solved by having the genome fold upon itself.

RNA viruses

(-) ssRNA

Filoviridae

Family Filoviridae
Virus classification
Group:	Group V ((−)ssRNA)
Order:	Mononegavirales
Family:	Filoviridae
Included genera
Genus "Cuevavirus" (proposed) Genus Ebolavirus Genus Marburgvirus

Filovirus are thread-like viruses. The envelope has a thread-like appearance and are studded with glycoproteins. Once inside the endosome, a glycoprotein is cleaved by cysteine proteases. This somehow allows the virus to bind to NPC1 which triggers fusion.

Ebolavirus

Genus Ebolavirus
Virus classification
Group:	Group V ((−)ssRNA)
Order:	Mononegavirales
Family:	Filoviridae
Genus:	Ebolavirus
Species
Bundibugyo ebolavirus Reston ebolavirus Sudan ebolavirus Taï Forest ebolavirus Zaire ebolavirus

Like the non-templated replication in mumps and measle virus, the GP gene in the Ebola virus genome also has an editing site. This gives rise to two glycoproteins - one which is non-structural and secreted (sGP) and one which becomes membrane embedded (GP). The GP will only be made when the GP gene is edited.

Paramyxoviridae

Paramyxovirus
Virus classification
Group:	Group V ((−)ssRNA)
Order:	Mononegavirales
Family:	Paramyxoviridae
Genera
Subfamily Paramyxovirinae    Avulavirus    Ferlavirus    Henipavirus    Morbillivirus    Respirovirus    Rubulavirus    TPMV-like viruses Subfamily Pneumovirinae    Pneumovirus    Metapneumovirus

Another mRNA synthesized from the replication of the genomic RNA is the P mRNA. In measles and mumps virus, some P mRNA have an extra base added during RNA synthesis, and this gives two different mRNA (and thus two different proteins) from one genomic RNA sequence. The RdRP replicates from the (-) genomic RNA strand; it will encounter a segment of repeated U/A's followed by G/C's. The mRNA gets transcribed up til the 2 G/C's and halts because of the U/A repeats. One of the G's (on the nascent strand) slips back one place creating a sort of bulge, and so the sequences downstream of this bulge will be frame-shifted by one base, meaning the polypeptide downstream will also be different. This occurs in about 10% of the time, and occurs at the editing site, and probably regulated by a stem-loop structure. This phenomenon is termed non-templated replication.

dsRNA

The (+) RNA in the dsRNA cannot be accessed by ribosomes, and so cannot be directly translated; thus dsRNAs must also come with its own polymerases. The polymerases uses the (-) strand to make (+) strand full-length complements, which can be translated to give proteins, or used to synthesize (-) strand, creating back the dsRNA duplex.

Reovirus

Reoviruses
Intact double-shelledRotavirus particles
Virus classification
Group:	Group III (dsRNA)
Order:	Unassigned
Family:	Reoviridae
subfamily
Sedoreovirinae Spinareovirinae

Reovirus have two concentric capsids. When it enters the cell through endosomes, the outer capsid is degraded via proteolysis. The inner capsid is much more hydrophobic and allows it to fuse with the endosomal membrane and be released into the cytoplasm. However, even in the cytoplasm, the reovirus do not release its dsRNA genome, instead, transcription occurs within the virus particle, and the mRNA are released though one of the five fold axis of symmetry to be translated in the cytoplasm. The mRNA gets translated and produce viral particles, each virion contains a single mRNA, which then gets transcribed to make the (-) strand.

Replication in reoviruses is conservative. In semi-conservative replication, the two strands of the viral genome is separated and each is made into a dsRNA. In conservative replication, only one strand is copied.

Retro-transcribing viruses

(+) ssRNA RT

There is only one family within this class - Retroviridae, and is the cause of human immunodeficiency virus (HIV) and Human T-lymphotropic virus.

The (+) ssRNA genome is not directly translated unlike ssRNA (+) viruses, but is instead retrotranscribed into a (-) DNA, which is duplexed. The dsDNA intermediate do not float around in the nucleus like other viruses, but instead integrates itself into the host DNA to become a permanent part of the host genome. The host is now a provirus (defined as a virus genome that is integrated into the DNA of a host cell) because the host now has all the genetic information to recreate the virus. The incorporated dsDNA then serves as the template for RNA production, producing mRNA to make proteins, as well as (+) ssRNA as replicated genome.

Viruses to look at

(in reverse order of prevalence in humans) HTLV-1, HDV, HIV, Adenovirus, GBV-C, KBHV, HGV, Papilloma, HBV, H3V-1, AAV, Polyoma BK, Polyoma JC, CMV, EBV, VZV, HHV-8, HHV-7, Anellovirus, ERV

History

Bacteriophages were studied.

It was not until 1949 when viruses were able to be studied in human tissues. John Franklin Enders, Thomas Huckle Weller and Frederick Chapman Robbins successfully propagated poliomyelitis viruses in primary human embryonic tissue cultures, for which they won The Nobel Prize in Physiology or Medicine in 1954.

Infectious cycle

Virus replication, or the infectious cycle, is the process by which a virus is replicated. In overview, the infectious cycle begins with the attachment of the virus onto a cell, entry into the cell, production of many copies of the viral genome and proteins, packaging of the genomes into particles, and the release of the particle to infect other cells.

Although the infectious cycle is often represented as a sequential process, it should be more realistically portrayed as a continuum.

Attachement and entry

The first stage of the infectious cycle is the attachment of the virus onto the cell surface. This attachment is facilitated by specific receptors on the cell surface. A cell is termed 'susceptible' if it displays functional receptors which is able to bind to a particular virus, this is regardless of whether it is able to support the whole viral replication process (this is termed 'permissive'). 'Resistant' cells do not contain a functional receptor.

Polio does not require the nucleus.

Attachment and entry of the virus into host cells is essential in the infectious cycle, because viruses are obligate intracellular parasites. Viruses cannot simply diffuse into the cell because they are too large to pass through pores on the plasma membranes.

This process is broken into roughly three steps - non-specific electrostatic interaction that leads to weak adhesion to the cell surface, specific receptor-ligand interaction, and transfer of genome into the cell.

Almost all viruses require receptors on the cell surface to infect the cell. The same receptor can bind to different viruses, and the same virus can bind to different receptors.

Attachment are often reciprocal. Influenza viruses expresses influenza haemagglutinin which binds specifically to sialic acids. Influenza A and B binds to N-acetylneuraminic acid, and influenza C binds to 9-O-acetyl-N-neuraminic acid. These sialic acids found in nature are almost terminal, and are used to cap the end of a glycoprotein. Viruses which infect humans preferentially bind to sialic acids in an α(2,6) linkage, and viruses which infect birds bind to sialic acids in a α(2,3) linkage. Humans have α(2,6) expressed on the upper and lower tracts of the lungs, but also have α(2,3) on the lower tracts of the lungs, and so humans are still suceptible to avian influenza viruses.

So haemagglutinin on influenza viruses binds to terminating sialic acids on glycoconjugates on the cell surface, but sialic acids on the influenza virus can also be bound by receptors on the cell surface. Envelope glycoprotein gp120 is a glycoprotein exposed on the surface of the HIV envelope; gp120 can bind to CD4 on surfaces of cells, especially helper T cells. The gp120-CD4 interaction leads to the fusion of the membranes (HIV is an envelope virus).

Movement of viruses inside the cell does not rely on simple diffusion. Taking into account the viscosity of the cytoplasm, viruses will take a relatively long time to diffuse across a cell, and it'd take even longer for it to find the right target (be it the nucleus or next to a ribosome); and so transport of viruses and viral components (genome, proteins etc) must be through cellular transport machinery. These include dynein/microtubule-mediated transport towards the nucleus, endocytosis, clathrin- or caveolin-mediated vesicle transport etc.

To ensure specificity of fusion, at least two recognition events usually must occur before the membranes are fused. Fusion usually require a fusion peptide on one membrane to be inserted into the other membrane; fusion peptides are usually shielded away fro fusion, until a signal (conformational change from a neighbouring protein) is present. Viruses favours this specificity because they must enter a cell which allows its genome to be replicated, otherwise its genome would be lost. This can be done either through binding of multiple ligand-receptor pairs, such as gp120/CD4 and HA/NeuAc. But even in these cases, the receptor for the ligand might bind, but does not directly cause the fusion peptide to insert into the membrane; but instead it causes a conformational change in the ligand which allows another receptor to bind more strongly. This subsequently binding causes the fusion peptide to insert. This mechanism ensures a specific binding using a two-hit mechanism.

For (+) RNA viruses, once the virus envelope and the plasma membrane fuse, the RNA is released into the cytoplasm, which can then make mRNA straight away (does not get translated).

Some viruses which enter the cell through the endocytic pathway. As the virus in endosomes matures, they are acidified. The lowered pH can cause a conformational change in the haemagluttinin which causes the fusion peptides to insert into the endosomal membrane, allowing the virus envelope to fuse with the endosomal membrane, releasing the viral content into the cytoplasm. There are three major types of fusion peptides - class II are usually α-helices perpendicular to the membrane, class II are usually β-sheets which lie parallel to the membrane, and class II are usually perpendicular to the membrane, forms trimers and contain a mix of α-helices and β-sheets.

Fusion is regulated - proteolytic cleavage is required to cleave a part of the protein to reveal class I fusion protein for fusion; class II fusion peptide activation usually relies on the cleavage of a second protein. pH also affects fusion.

Laboratory techniques

Discovery of new viruses

Koch's postulates of disease.

Viruses causes diseases. Therefore, if the novel suspected virus particle is able to cause disease, then it is a new virus, or a new strain of a virus. The establishment of causality has traditionally been determined by how well the suspected virus conform to Koch's postulates:

1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

However, many of the viruses (Hepatitis C, HHV-8, HPV) cannot be grown and were discovered before they can be grown. Nowadays, it is not the microorganism which is measured, but the viral-specific nucleic acid.

Virus cultivation

Embryonic inoculation

One of the first methods of growing viruses was inoculating the virus in embryonic eggs, such as the chicken egg. Different viruses are inoculated at different locations. Herpes simplex virus, poxvirus and the Rous sarcoma virus are introduced into the chorioallantoic membrane of the egg; influenza virus and mumps virus can be introduced in the amniotic or allantoic cavities. Herpes simplex virus can also be injected into the yolk sac. Newcastle disease virus and avian adenoviruses are introduced into the allantoic cavity.

Nowadays, only allantoic inoculation of influenza virus are still performed today, and is used to produce the influenza vaccines. Because these vaccines are produced in egg, people with allergies to eggs or feathers should not be injected with this type of influenza vaccines.

Cell culture

Nowadays, most viruses are cultivated in cell culture. There were two type of cell cultures - that which uses primary cells, and those that uses immortalized cell lines. Primary cells are derived from tissues directly, separated and grown in culture for a limited time, before it dies off; primary human foreskin fibroblast is commonly used in this manner. Immortalized cell lines such as the mouse fibroblast cell line 3T3, and human epithelial cell line HeLa, are immortalized cell lines which are able to be grown and replicated indefinitely; however, they tend to have abnormalities such as abnormal number of chromosomes and/or nuclei, and thus is not ideal platform to propagate vaccines with.

Diploid cell strains such as WI-38 were developed to contain the correct number of chromosomes and is useful for vaccine production; they do die off eventually but do live longer than primary cells.

Quantification of viruses

Cytopathic effect

The growth of the virus can be measured by the cytopathic effect (CPE) - alterations of the shapes and behavior of the cells which are caused by the virus. For example, in HeLa cells, virus causes the cells to round up and detached, before dying. Other common cytopathic effects include morphological ones, such as the formation of syncytia (the fusion of cells), pyknosis (nuclear shrinkage), proliferation of nuclear membrane, vacuiles in cytoplasm, margination and breaking of chromosomes, and rounding up and detachement of cultured cells, as well as the appearance of inclusion bodies. The types of CPE observed depends heavily on the type of viruses that is infecting the cell.

The CPE is not a good indication of viral propagation, as it is hard to distinguish its effect from those caused by apoptotic signals arising from overcrowding and other viral-unrelated causes.

Plaque assay & Endpoint dilution assay

The first infectivity assay is the plaque assay, developed in the 1930s. Viruses was mixed with bacterial culture, and spread on an agar plate. The bacteria forms a monolayer on the plate, but are absent in areas where the virus are propagating. Viruses (must be lytic) destroy the cells it is infecting and creates cleared areas, or plaques. Each plaque is assumed to be caused by one virus, and the number of viruses can be calculated this way. The assay is usually performed as a series of dilutions (usually 10-fold) and the most useful plates were used for calculation. The virus is prevented from diffusing to other parts of the plate by covering a semi-solid overlay (e.g. agarose, carboxymethyl cellulose) on top of the plate. This overlay can be removed after incubation to allow the underlying cells to be stained.

The quantification of viruses are measured as plaque-forming units (PFUs). Typically for most bacteriophages, one viral particle produce one plaque, and so the number of plaques roughly equates to the number of virus particles. However, animal viruses often require more than one particle to form a plaque, and so it's harder to determine the exact number of viral particles. he particle/PFU ration can range from 1 to 10000. The cause of these variability may be due to damage to the particles, mutations specific to each viral particle, as well as variability in the infectious cycle.

The plaque assay was first performed in bacteriophages, and then in 1952, Renato Dulbecco performed the plaque assay with animal viruses on monolayer tissue cultures of animal cells. Plaque formation has been documented in real time.

Some viruses do not form plaques on any type of cells, and so another technique - the endpoint dilution assay - is used instead. It measures cell killing using different concentrations of viruses. The 50% Tissue Culture Infective Dose (TCID₅₀) is defined as the concentration in which half the wells with cells infected at that concentration is dead.

Haemagglutination assay

The haemagglutination assay (HA) is first conducted by George K Hirst in 1941-1942.Influenza viruses binds to sialic-acid-terminating glycan structures found on cell surfaces. Erythrocytes contain these NeuAc-terminating glycans and can be bound by influenza viruses. This binding causes the erythrocytes to aggregate as one virus can bind to multiple erythrocytes. The dilution in which aggregates are no longer observed (the HA titre) gives an indication to the concentration of the virus.

Measurement of viral enzymes

Some animal virus particles contain nucleic acid polymerases and other enzymes, and their activity can be measured by permeabilizing the particles and incubating them with the substrate of the enzyme. Products, proportional in concentration to the concentration of the enzyme, is produced and can be measured. The amount of enzymes is then assumed to be proportional to the number of viral particles.

This is often done for retroviruses, many of which do not transform cells or form plaques.

Immunostaining

Fluorescently-tagged antibodies against surface viral proteins can bind to the virus and can be visualized using fluorescence microscopy. Other ways of visualizing is also possible.

Immunoblotting

Viral proteins can be separated on a polyacrylamide gel and transferred onto a nitrocellulose membrane and blotted with labelled antibodies, which can be detected subsequently. This is known as Western blotting.

A similar technique can be done with antibodies against viral DNA (Southern blot) or RNA (Northern blot).

Enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a technique whereby a 'capture antibody' attached to a solid support is used to bind to viral antigens in the sample. The surface is washed before a second fluorescently-labelled labelled antibody is used to bind to the viral antigen (through a different epitope). The level of fluorescence is proportional to the abundance of the antigen in the sample.

This can also be used in a clinical setting to quantify the concentration of antibodies against an antigen, whereby the antigen is attached to the solid support, and the secondary antibody is one which recognizes the antibody in the sample, often through binding to its constant domains.

Quantitative polymerase chain reaction (qPCR)

Specific primers that flanks the viral gene is introduced along with purified DNA derived from cell lysates. These are incubated in a special buffer along with a heat-resistant polymerase, nucleic acids, and a fluorescent dye such as SYBR Green I (SG). The primers bind to either side of the gene, and allow the polymerase to polymerize from it, creating multiple copies of the gene. SG only binds to double-stranded DNA and is an indication of the quantity of the amplified gene. The higher the fluorescence signal, the higher the original concentration of the viral gene, and thus, can be assumed, a higher concentration of the virus.

Induce mutation

Nitrous acid, hydroxylamine and alkylating agents directly modify the viral DNA to change is constituent sequence. Base analogs, intercalating agents, UV radiation can cause mutations during replication.

Prevention against disease

Before any defenses are employed by the host, a virus must first encounter the host. Unlike other microorganisms, a virus particle (virion) has not means of locomotion, and must rely on diffusion-limited processes. Furthermore, the external environment in which virions are often exposed can be harsh with heavy UV radiation, drying, extremem pHs etc.

Once the virus encounters the host, it must gain entry into cells. The most prevalent feature of a host is the skin. The skin is covered with dead cells which are shed regularly, and there are sebaceous gland on the skin which secretes acids that keep the skin at a low pH, an environment which is harsh for viruses. Mucus and the extracellular matrix are other physical components which keeps the virus away from cells. The host most likely also has an immune system which recognizes antigens on the surface of the virion and directs an immune response targeting it.

Viral genome

The nucleic acid genome carries all the information required for the replication and spread of virions. There are seven different viral genome types, made up of either DNA or RNA, and are classified using the Baltimore classification based on the type of viral genome. DNA viruses include dsDNA (I) and ssDNA (II). RNA viruses include dsRNA (III), ssRNA (+) (IV) and ssRNA (-) (V). Retro-transcribing viruses incude ssRNA (RT) (VI) and dsDNA (RT) (VII). Regardless of viral types, all must make mRNA, because all must make proteins. However, not all (+) strand RNA are mRNAs. Different viral types have different sets of unique polymerase, primers, template and termination requirements which synthesis the mRNA. All viruses within a family follows the same/similar strategy for making mRNA.

The structures of the genome are diverse; they can be linear or circular, segmented (many strands), gapped, single-stranded (+ or -, with + meaning sense strand, which can be translated straight away) or doubled stranded. Some have the ends of a dsDNA crosslinked, some have covalently attached proteins, and some have DNA covalently attached RNA.

The genomic information in any virus doe not encode for a complete protein synthesis machinery, and must rely, at least in parts, on the host machinery. Commonly, the viral genomes make mRNA which are translated by the host ribosomes. There are also no genes encoding for proteins involved in energy production or membrane synthesis, which are both required for the infectious cycle, and these must come from the host. Viral genomes also has non-coding regions.

The viral genomes do not have classical centromeres or telomeres.

Single-stranded DNA virus genomes must first become dsDNA so that mRNA can be transcribed from it

Virus with a gapped dsDNA genome must be repaired by the host machinery before it can transcribe RNA.

Most cells in the body are terminally differentiated, and have permanently entered the G₀ phase of the cell cycle, and thus are incapable of replicating DNA. When DNA viruses enter these cells...

The topology of a virus genome is critical.

RNA viruses are error prone in their replication, and usually have 1 mutation event every 10⁴ to 10⁵; DNA viruses are more stable and have 1 error per 10⁸ to 10⁹ events.

dsDNA

There are 22 families of viruses with a dsDNA genomes, which have either linear or circular genomes. To make the mRNA, the DNA must be double-stranded and is copied from the (-) strand, which is complementary to the eventual mRNA.

Gapped DNA=

Hepadnaviruses, hepatitis B

Gapped DNA are made up of circular dsDNA. There is a protein covalently atached to the 5'-end of one strand, and a short RNA is covalently attached to the 5'-end of the other strand. One strand is complete whereas the other strand is about half complete.

To make the mRNA, the covalently-attached protein and mRNA must be removed, and the gap from the half-complete DNA strand must be filled in to create a double-stranded DNA to make mRNA.

The virus also transcribes RNA from the DNA which is not mRNA and not used to translate into proteins; rather, it is used to make (-) DNA using a reverse transcriptase, which is duplexed into dsDNA to replicate the viral genome. The reason behind the RNA intermediate during DNA replication is unclear.

ssDNA

Circoviridae (TT virus - ubiquitous human virus), Parvoviridae (B19 parvovirus - fifth disease)

There are 5 viral families in this class. The ssDNA must first be duplexed to make dsDNA, in order to allow for transcription. ssDNA viruses have small genomes and do not encode DNA polymerase, and thus must rely on host DNA polymerase. As host DNA polymerases are located in the nucleus, the viral genome must travel to the nucleus as part of its infectious cycle. ssDNA viruses also do not encode for RNA polymerases.

RNA genomes

There are no RNA-dependent RNA polymerases in host cells, and thus viruses with RNA genomes must either retro-transcribe DNA from RNA (as it is with the ssRNA retro-transcribing viruses) or encode for a RNA-dependent RNA polymerase.

dsRNA

There are 7 families of dsRNA viruses, and many of these are segmented - Reoviridaw have 10-12 segments of dsRNA, Birnaviridae have 2 segments. The dsRNA cannot be translated, and must rely on the RNA-dependenet RNA polymerase to transcribe RNA which is used to make mRNA, as well as to make more copies of the genome.

ssRNA (+)

There are 22 viral families in the ssRNA class, 8 of which can infect mammals. The ssRNA genome are directly translated into proteins by the host ribosome. Some mRNAs and copies of the genome are first transcribed into (-) RNA, which functions only as a template to reproduce the ssRNA (+) genome.

ssRNA (-)

There are seven families in this class. The (-) strand RNA cannot be translated, and is used as a template to copy into a (+) strand using a viral-encoded RNA-dependent RNA polymerase. The (+) strand can then be used to make proteins, or be used as a template to make (-) RNAs, which is packaged back into the virus for its replication.

Some of these viruses (Arenaviridae, Bunyaviridae) are ambisense, meaning they contain both a (+) and (-) strand.

Segmented genome

Viruses with segmented genomes mean that if two different viruses of the same family infect the same cell, the segments can be mixed (reassortment) between the parental viruses, and the replicated viruses will have a mixture of these segments. This genetic mixing (reassortment) is also observed with recombination in host cells, but reassortment in viruses occurs more frequently because you do not need breakage and religation of dsDNA, only mixing.

Reassortment is also the basis of transmission of viruses across different species. An avian virus with a segmented genome can mix its genome with that of a human virus of the same family, and may give it a receptor (haemaglutinin in influenza) that allows it to bind to a human-specific ligand (α2,6 is bound by human haemaglutinin). This sort of reassortment usually occurs in an intermediate host, which can host both types of viruses. For influenza, the pig can host both avian and human influenza, and is a common exchange site of avian and human influenza genomes.

Genome structures

The viral genome is likely to take on secondary conformations and have specific configurations that are essential to its function.

Structure

The viral proteins have different roles - in short, for protection and delivery the genome.

Virions are metastable structures, which are not in their lowest free energy conformation. They are able to persist in this state for a long time until they encounter a trigger, which occurs during attachment and entry. This metastable allows the viral protein to protect the genome in its stable state, but allow it to come apart quickly in its unstable state, in order to release the genome into the host cell.

Some viruses have replication and transcription of the viral genome occuring within the virion inside the cell, and the viral structure do not have to completely break apart, but only needs to open up. For example, retroviruses have the RNA retrotranscribed inside the virion, and Reoviruses (dsRNA) have the replication and mRNA synthesis inside the virion.

The virion structure consist of symmetrical arrangements of many of a few identical proteins into regular 3D structures, with the proteins held together by identical non-covalent interactions between identical subunits, which are broken apart to allow the genome to come out of the virion. All interactions of the subunits are identical, and are either head-to-head or tail-to-tail.

When a mixture of purified TMV RNA and coat protein, they were assembled spontaneously.

The capsid proteins form a protective and stable shell which recognize specifically the viral and not host genome, and packages them. In some viruses, there is a membrane envelop, which interacts with the capsid proteins. This envelop is a lipid bilayer derived from the host cell; because the virus cannot make lipids itself, it must depend on the host cell for this envelop. Curiously, plant viruses are rarely enveloped. Many capsid proteins can self-assemble into virus-like particles (VLPs).

There are basically two different structures taken by viruses - rod-shaped viruses and icosahedron (20 equilateral triangular faces, 12 vertices) viruses. Rod-shaped viruses (such as TMV) have coat proteins that interact with each other, as well as the viral genome, using identical interactions. The rod is actually coat proteins wrapped around the RNA genome in a helical arrangement, similar to a coil arrangement. Most rod-like viruses have envelops, with the obvious exception of TMV. These capsid proteins are often termed nucleocapsids, because it interacts with the genome itself. Other viruses have both capsids and nucleocapsids.

All icosahedron proteins proteins have a defined number of proteins; multiples of 60s are common. The capsid proteins are aoften arranged as hexamers and pentamers. The overall number of capsid proteins follow a discreet allowable values of triangulation (T) numbers (T=1 means the virus have 60 subunits).

The majority of bacteriophages have a tail which extends from the head and is used for attachment to the host cell, penetration of the envelop, and once a hole is made in the host membrane, the DNA is injected into the host under high pressure.

Envelope

Envelope is a lipid bilayer derived from the host cell; because the virus cannot make lipids itself, it must depend on the host cell for this envelop. Curiously, plant viruses are rarely enveloped.

Envelopes usually have integral glycoproteins on them. The outer domain (ectodomain) is used for attachment, antigenic sites, fusions and other functions, whereas the internal domain is required for multimerization.

The inner domain can interact in different ways. It can directly contact with the capsid or nucleocapsid; in these instances, the symmetry of the underlying capsid will be reflected in the arrangement of the glycoprotein; these are structured envelopes. The glycoprotein can also interact indirectly with the capsid through a matrix protein (M protein), or through multilayer of internal proteins; these are unstructured envelopes.

HIV

In birds, the sialic acids presented on avian cells are α2,3 linkages, and those on humans are α2,6 linkages. An influenza virus which binds to α2,3 do not bind to α2,6 linked NeuAc. For an avian virus to infect humans, a mutation must be made that allow the haemaglutinin molecule to bind to α2,6; these mutations occur from nucleotide mutations. Difference between avian and human H3, there is only two nucleotide difference, represented as two amino acid mutations.

Ferret is the closest human model used for influenza virus. They have the same receptors as humans.

Clinical Relevance

Gene therapy

Many diseases are caused by defect(s) in a single gene which means the gene is not expressed and the organism lacks that protein. Gene therapy is a method to introduce, through viral transfection, a copy of the gene embedded in the viral genome (called a vector), which when transcribed, allows the missing gene to be expressed, and thus compensates for the lack of it in the host. Common vectors include adenoviruses and retroviruses, the former do not incorporate into genome and acts only transciently.

Haemophilia A is caused by Factor VII deficiency, haemophilia B is caused by factor IX deficiency, familial hypercholesterolemia is caused by deficiency in the low-density lipoprotein receptor, cystic fibrosis is caused by defects in the α- or β globin gene, Gaucher's disease is caused by defects in glucocerebrosidase, Duchenne muscular dystrophy is caused by the lack of production of dystrophin.

Influenza

Influenza A contains a M2 protein, a proton-selectiveion channelprotein. It is made up of a homodimer of M2 subunits, and stabilized by two disulphide bonds. It is activated in low pH (usually in the endosomal environment) through the cleavage of the disulphide bonds. This causes protons to enter into the virion. This process is not involved in the fusion of the viral/endosomal membrane, but rather dissociates the viral matrix protein M1 from the ribonucleoprotein RNP. This means the nucleic acid genome from the viral membrane, so when fusion occurs, the genome is released into the cytoplasm and not remain attached to the membrane.

Dengue

The dengue virus have mostly class II fusion peptides which lies on the surface of the virion. Upon uptake into endosomes by endocytosis, the lowered pH causes these fusion peptides to stand up and insert into the endosomal membrane, and eventually causes fusion.

Adenovirus

Adenoviruses
Transmission electron micrograph of two adenovirus particles
Virus classification
Group:	Group I (dsDNA)
Family:	Adenoviridae
Genera
Atadenovirus Aviadenovirus Ichtadenovirus Mastadenovirus Siadenovirus

The adenovirus contains protein VI in its capsid. This is usually hidden away. As the endosome containing the adenovirus becomes acidified, the adenovirus starts to disintegrate, releasing protein VI, which can now insert into the endosomal membrane and lyse the membrane, releasing the virion as well as other contents of the endosome.

The virus then travels down microtubules, facilitated by dynein motors, onto the nuclear pore complexes, where uncoating and insertion of the viral genome into the nucleus occurs.

Poliovirus

Poliovirus
TEM micrograph of poliovirusvirions. Scale bar, 50 nm.
Virus classification
Group:	Group IV ((+)ssRNA)
Order:	Picornavirales
Family:	Picornaviridae
Genus:	Enterovirus
Type species
Human enterovirus C
Species
Human enterovirus C

The binding of the receptor for Polio is sufficient enough for the virus to make a pore by inserting a lipid from the virus into the endosomal membrane. The viral lipid are linked to the virus, and forms a pore to allow the viral components to be released.

The genomic of Polio virus resembles mRNA in many ways - polyadenylated 3' end, 5' and 3' untranslated regions (UTR). In the 5' end of an mRNA, there would be a capping protein. In the viral genomic RNA, this capping protein is instead a different protein; this protein is a protein primer.

The whole genome is translated in one go, creating a massive polypeptide. This polypeptide contains within it many different proteins, which will eventually form the capsid, proteases and RNA synthesis machinery, all linked together. One of the proteins which emerges from this translation are proteases, such as 2A^pro and 3A^pro, which cleaves the massive polypeptide chain into many pieces. These proteins include VPg, which is the protein primer for the viral genome.

RNA have secondary structures; stem-loop structures, cloerleaf, pseudo-knot, cis-acting RNA elements are common. These secondary structures might be involved in the specificity of the RdRP, to prevent it from replicating cellular RNA, which lack these secondary structure.

VPg associate with complexes associated with the Cre structure, and gets two uridine phosphate added onto the protein, and uses it to prime the strand.

Coxsackievirus

Coxsackievirus
Virus classification
Group:	Group IV ((+)ssRNA)
Family:	Picornaviridae
Genus:	Enterovirus
Species:	Human enterovirus A, B and C

Coxsackieviruses initiate infection at the epithelial surfaces.

Coxsackievirus group B viruses requires binding of two receptors - Decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) - in order to infect cells. DAF is displayed on the surface of epithelial cells and can bind to the virus; CAR, however, is a component of tight junctions and not accessible to the virus. In fact, DAF is present on the apical side of epithelial cells; when DAF is bound, it signals for loosening the cytoskeleton between the cells, loosening the tight junction, allowing the virus to bind to CAR.

Questions

must a retrovirus genome be integrated into the genome BEFORE further RNAs are transcribed from it?

RNA-depedent RNA polymerase

Replicase (an enzyme that copies viral RNA to produce genome)

Transcriptase (enzyme that produces mRNA)

The RNA template tends to differ between different classes of RNA viruses. (-) ssRNA viruses have RNA genomes coated with proteins; these proteins include nucleoproteins and polymerases, and is more like the ribonuclear protein complexes. (+) ssRNA virus genomes are usually naked, ready to be translated by host machinery (retroviruses do not get translated straight away). Retrovirus genome are coated with proteins that helps with the retrotranscription. Coronavirus is a (+) RNA virus that also has a genome that come coated with proteins, but its function is unclear; the large size of its RNA genome might play a role. dsRNA genomes usually come with an RNA polymerase, since the host lack this enzyme.

RNA-dependent RNA polymerases may initiate polymerization with or without (de novo) a primer, and operates in a 5' to 3' direction (the template is read from a 3' to 5' direction). All polymerases have common motifs, (+) strand RNA polymerases have a conserved sequence of Gly-Asp-Asp; Asp-Asp are found in RT, segmented (-) strand polymerase; Gly-Asp-Asn is found in non-segmented (-) strand polymerase; Ala-Asp-Asn are found in birnaviral polymerases.

All (+) strand viruses induce the formation of vesicles and dissolves the ER and Golgi. RNA synthesis occurs on the surface of these vesicles; this is probably so that they can concentrate the machinery all in one location. Some of the (+) RNA are immediately translated to give viral proteins, while some move onto these membranous vesicles to be a template to make (-) RNA whole genome copies, which are further transcribed back to the (+) RNA genome, which can be translated again, but also ready to be packaged by the viral proteins, which was made in parallel to RNA 'replication'.

Alphaviruses

Alphavirus
Virus classification
Group:	Group IV ((+)ssRNA)
Order:	Unassigned
Family:	Togaviridae
Genus:	Alphavirus
Type species
Sindbis virus
Species
Aura virus Babanki virus Barmah Forest virus Bebaru virus Cabassou virus Chikungunya virus Eastern equine encephalitis virus Everglades virus Fort Morgan virus Getah virus Highlands J virus Kyzylagach virus Mayaro virus Me Tri virus Middelburg virus Mosso das Pedras virus Mucambo virus Ndumu virus O'nyong-nyong virus Pixuna virus Rio Negro virus Ross River virus Salmon pancreas disease virus Semliki Forest virus Sindbis virus Southern elephant seal virus Tonate virus Trocara virus Una virus Venezuelan equine encephalitis virus Western equine encephalitis virus Whataroa virus

Alphaviruses are (+) ssRNA viruses, but unlike Poliovirus, the genomic RNA does not get translated much, but most are used as a template to make (-) full-length complement, which is then used to make mRNA and the genomic RNA. Unlike Poliovirus, the RNA genome 5' end is capped and not primed by VPg.

The genomic RNA gets translated by ribosomes, translate the genome as a whole, and gives a long polypeptide (P1234) which is proteolytically cleaved to evolve viral RNA polymerases (nsP1/2/3/4), which then go on to make (+/-) RNA. The (-) strand is then used to synthesis mRNAs, which encodes for capsids and other proteins. This additional mRNA step is required because the RNA of the alphavirus contain a stop codon, which stops the RNA from being translated downstream of the stop codon; thus mRNA from downstream of the stop codon must be initiated downstream of the stop codon.

(-) strand RNA virus

The (-) strand usually comes along with attached polymerase, which makes individual mRNAs that encode for all the individual proteins. One of the proteins produced includes a protein called N. Usually, the RdRP will stop after each gene due to the presence of intergenic regions (ig), N proteins coat these regions and antagonize them. The N protein is absent at the beginning of infection, and so all the synthesis is mRNAs; when the level of N proteins rise, they ensure the RdRP keeps transcribing the entire genome and not stop.