Lecture 2 - Virology basics pt1

Definitions & Virology vocabulary

Vocabulary

Virions

It is the physical particle in the extra-cellular space (medium)

Viral genome

It is a molecule of DNA or RNA, depending on the virus family

Capsid (C) or nucleocapsid proteins (N or NP)

They are the proteins which cover viral genome

Nucleocapsid complex or nucleocapsid

It is the combination of the capsid and the genome

Envelope

It is the lipidic membrane surrounding the virion

Viral proteins

They are proteins encoded by the viral genome

Structural proteins

They are the viral proteins that constitute the virion

Non-structural proteins

They are viral proteins that are not included in virions, meaning they are in the cell or the extra-cellular matrix not inside the virion.

Surface glycoproteins

They are glycoproteins incorporated in the virion’s envelope

Viral structure

Viral particle are generally composed of :

• nucleic acids
• proteins
• lipids

They are two types of viruses : naked virus (also called non-enveloped viruses) and enveloped viruses. \n The enveloppe is a lipid bilayer membraneusually taken from a cell. In this membrane, there are surface glycoproteins. \n Inside the viral particule, there is only the nucleocapsid (genome + proteins). Although, for enveloped viruses, there is also other structural proteins inside the enveloppe as well as in the enveloppe (surface glycoproteins)

Exception : Virions of family Arenaviridae include cellular ribosomes

Viral proteins can be found in the virion (structural proteins), inside the cell or outside the cell. \n The enveloppe can be gained from several organelles :

• nuclei membrane ??
• endoplasmic reticulum
• the golgi apparatus
• the trans golgi network ??
• multivesicular body ??
• vacuole ??
• cytoplasmic membrane

Virus size are usually from 30 nm to 1000 nm. In comparaison, bacteria have a size range of 100 to 10 000 nm and eukaryotic cells of 4 000 to 40 000 nm. Consequently, most of them aren't visible by light microscope but they are visible with an electron microscope. They also come in a variety of shapes. \n

They are obligate intracellular parasites, meaning they require cells to replicate.

For RNA viruses, the mean genome size is of 9 kb with a range of 1.7 kb (Hepatitis delta virus) to 30 kb (Coronavirus). \n ForDNA viruses, the size of genome varies greatly from less than 2 kb (Circoviridae) to the largest viral genome 2.7 Mbp (Pandoravirus).

Basic methods to study viruses

Virus are cultivated in cells or in organisms like animals, plants or chicken eggs. \n Different factors need to be taken into account for viral cultivation ; the type of cells (blood, liver, skin…), the cell lines (the ATCC collection), if there is susceptible animals, if the cells are susceptible, if they grow in suspension or if they are adherent and lastly the percent of confluency (meaning the % of the surface covered by adherent cells). \n If the confluency is too high, the cells stop growing thus hindering virus replication. Usually a confluency between 30 and 50% is best. \n

Cytopathic effects

Viruses always inhibit or activate something in cells when they replicate. Any detectable changes in the host cells is called cytopathic effect or CPE \n It can be :

• Morphological changes of cells (see image below)

• Cell death by Apoptosis or Necrosis or Lysis

• Indefinite growth :

  • Pock formation on the chorioallantoic membrane of eggs
  • Transformed cells growth SV40

• Formation of multinuclear cells (syncytia)

• Observation of viral inclusions bodies within infected cells

  

For example, the cytopathic effect of Ebola virus on Vero E6 cells is cell rounding and detachment. We can compare the mock condition with all attached cells and the most cytotoxic condition Ebov-8A. \n Adherent cells have a prism-like shape that is recognizable and different from the rounded shape. \n

A second CPE is syncytia, meaning the formation of giant cell with multiple nuclei after fusion of cells expressing viral surface glycoproteins and neighboring cells expressing viral receptors. Syncytia has been noticed for Nipah virus, RSV or Measles for example.

A third CPE is pock formation, meaning a visible foci or lesions on the chorioallantoic membrane of the developing chick embryo. \n

A fourth CPE is the transformation and loss of contact inhibition for growth. It is observable in petri dish, because the cells will multiply in only some points. \n

A fifth CPE is inclusion bodies. It has been noticed that for rabies virus, inclusion bodies are viral RNA replication sites. \n

Virus titration

Quantitative Analysis

Biological Titration

There is biological titration, that uses virus ability to infect cells (infectivity). It can only count infectious unit. If a thousand particles are needed to get one infected cell, a thousand particles will be counted as one infectious unit.

Limited dilutions can be used with plaque assay. The lowest dilution showing a CPE on cells is representative of the number of infectious unit in the virus stock.

1. Plaque assay was developed in early 1900 by d’Herelle for bacteriophages ; in 1953 for animal viruses by Dulbecco and Vogt.

It allows the count of plaque forming unit or pfu. Plaques are zones of dead cells caused by virus, usually visible by the naked eye. \n \n Method : Cover the cells with culture medium containing low melting Agar or 2% Methylcellulose or 1% Avicel (polymer beads). The coverage will prevent virus distribution in the medium, the virus will spread only to the neighboring cells. Staining of live cells will allow to visualize the plaques \n \n One PFU unit is different from one viral particle. The number of virion needed depend on several factors like mutations, inactivated virions, low susceptibility of cells etc

2. In case of transformation and formation of cell’s foci, focus assay is used.

The foci can be counted by eyes or by microscopy. Although, they sometimes need to be revealed by staining.

Both foci and plaques represent areas of infected cells

3. Another methods that can be used with limited dilution is the calculation of pocks on chorioallantoic membranes on embryonic eggs.

The endpoint of these methods

TCID50 or 50% Tissue Culture Infectious Dose \n It is the measure of the infectious virus titer. It correspond to the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells

LD50 or median lethal dose \n It is the dose required to kill half the members of a tested population.

Those methods are calculated by diluting the virus stock until it result in 0% infection then calculating the dilution giving 50% of replicate inoculations.

Physical and chemical quantification methods

There is also physical and chemical methods of virus quantification. These methods count the number of viral particle.

1. Particles count by electron microscopy

A method used is direct particle count(relative). \n For that negative staining technique with phosphotungstat contrasting is used. Beads of known concentration are added. Consequently, the beads and the virus particles are counted. Then, viral concentration is determined thanks to the beads concentration. Usually, about 10^7 beads per ml are needed. For virus concentration, we use ultracentrifugation.

2. Particles count: qPCR (count the number of genome copies).

   

Often difficult due to contaminations by virus specific mRNAs. It is also important to note that one genome can have multiple DNA segment. So we need to know how many genomes per virion. Other problems could come from : the efficiency of RNA/DNA extraction, defective particles, reverse transcription for RNA viruses \n cf : lessons on qPCR

3. Viral protein: Hemagglutination, ELISA, WB, neutralization assay

It shows the number of proteins, so the counting is relative to how many proteins each particle has. You need to know the number of proteins per particle for this. \n It can be done using protein patterns (gel, Coomassie, Silver) or antigen detections (WB, IF) \n Those methods are mostly used for comparing different strains, not for accurate quantification

(PAAG = polyacrylamide gel electrophoresis)

This western blot shows the apparition of viral proteins each day post infection (dpi).

When there is enough viral particles, they crosslink with red blood cells. Cross-linked blood cells sediments and cover the bottom of the well. When there isn't enough particle, they sediment in the well's center. This phenomenon is visible. Virus titer is counted as last virus dilution revealing the effect of hemagglutination

Those two methods combine other methods with anti-viral antibodies. It allows to determinate the concentration of serum needed to bind all the viral particles. In those case, the limiting dilution is when you observe no effect from the viral particle because they are bound to antibodies.

Multiplicity Of Infection or MOI

Infection unit

Number of infection units taken per one cell \n They are usually counted as equal to pfu.

MOI

Ratio of the number of virus particles to the number of target cells present in a defined space \n If two virus need to be compared, their MOI needs to be similar. \n A MOI of 1 means that their is one infectious unit per cell, which means that in theory there is a 100% of infection.

Single cycle infection

Almost all cells are infected by the initial inoculum. It correspond to a high MOI. \n For exemple, one could use a MOI of 2; if TCID50 method was used for virus titration

Multicycle infection

Only a low number of cells are infected by the inoculum. Several rounds of infection needed to pass for infection of majority of cells. Usually one will use low multiplicity of infection, as example MOI of 0,01; approximately 1 from 100 cells is infected

In practice, plague forming unit assay and TCID50 are most common.

Virus growth

Eclipse periodimmediately follows the penetration of viral particles into the host cell. No free virions to detect outside of cells. Virus need time for replication. \n High MOIVirus release and no more cells to infect. \n Low MOISeveral waves of virus release. Non-synchronized infection often prevent to observe the waves. \n

The entry phase. \n Recognition and attachment of the viral particle to cell, and the internalization and release of the viral genetic material within the host cell. \n The biosynthesis phase. \n Production of virus specific messenger RNA (mRNAs) – transcription, synthesis of viral proteins and the replication of the viral genetic material. \n The exit phase. \n Assembly of new viral particles and their release from the host cell.

Here the virus need substantial time for initial virus assembly – replication of viral genome, transcription, protein synthesis and assembly of viral particles.