Bacteriophages, Influenza, and Viral Study (PPT 3)

What is candling? (hint: involves an egg)

- A method used to observe the inside of an egg (and get the virus into the proper site) by shining a bright light source behind the egg

What is reassortment?

- A process that describes the switching of viral RNA (gene) segments in cells infected with two different influenza viruses (NOTE: Reassortment of the genome segments of two differing influenza strains has the potential to vastly increase the diversity of circulating influenza viruses) (FUN FACT: The Swine Flu of 2009 (or, H1N1) is a QUADRUPLE reassortment, containing RNA from two different pig influenza viruses, a human influenza virus and a bird influenza virus!)

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Adsorption (1)

- Attaches to surface of host; driven by the tail fibers and host cell receptors

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Synthesis (or Replication) (3)

- Duplication of phage components; replication of virus genetic material

Why are chicken eggs one of the best options for when we can't grow viruses in cells?

- Easy to get, and (relatively) cheap - Fertilized eggs contain a ton of rapidly dividing cells, growing in a naturally sterile environment containing lots of nutrients - And, once a chick starts growing, it offers multiple sites of inoculation, so we can study how viruses grow in different cells, and how they can potentially move between cells (NOTE: eggs are used to produce viral particles to study, and to develop vaccines to protect folks against viruses - purdy cool, y'all!)

General Influenza (Flu) Basics

- Flu viruses are part of the Orthomyxoviridae family of viruses (which includes: influenza A, influenza B, and influenza C) - Orthomyxoviridae viruses have enveloped nucleocapsids containing a genome composed of several (6-8) linear RNA strands - One of the BIGGEST viral killers

How can vaccines be produced?

- Killed whole cells - Inactivated viruses - Attenuated (live, but weakened) cells or infectious viruses

Influenza Spike: Hemagglutinin

- Main Role: to pull the viral membrane close to the host membrane and help fuse the two membranes so the viral genome (several strands of RNA) can get into the host cell - Hemagglutinin binds to chains of sugars (glycans) on host cells. Because there are multiple hemagglutinin spikes on its surface, a single virus can bind multiple cells. (NOTE: The name hemagglutinin comes from the ability of flu viruses to sometimes bind to and clump together (i.e. agglutinate) red blood cells)

So, how do we stop pandemics (ex. Smallpox, Influenza, etc.)?

- NOT an easy thing to do, as we have seen with COVID-19 - But, in the past, VACCINATION has been very successful in decreasing the incidences of many diseases, including: Smallpox (eradicated in 1980), Polio, and Diptheria.

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Assembly (4)

- New virions are assembled

Structure of Bacteriophage

- Nucleocapsid (capsid head) - Tail Fibers (the "spikes of bacteriophages"): recognize and bind specific proteins on the bacterial surface - A Base Plate (at end of the helical sheath): forms a place of attachment for tail pins - Tail Pins: help to anchor the virus to the bacteria

The Lysogenic State

- Some bacteriophages can insert their own genome into the genome of its host, and not go through the whole life cycle, this is called "the lysogenic state" (similar to latent state for some animal viruses) - The cause of lysogenic conversion (the transfer of new traits to bacteria)

Influenza A (tends to cause most misery in humans)

- Strains of influenza A are named for two of their genes that help them get into (and then out of) host cells - The first gene contains the recipe for a protein called hemagglutinin, which is the influenza spike. (Remember: viral spikes are proteins that allow the virus to dock onto the host cell)

After hemagglutinin helps fuse the two membranes (the viral membrane & host membrane) and the viral genome (several strands of RNA) gets INSIDE the host cell, what happens?

- The RNA genome takes over the host cell to make lots of copies of the flu virus (NOTE: One of the proteins encoded within the flu RNA genome is called neuraminidase. This protein becomes important when the new flu particles leave the host) (EXTRA: At least sixteen different variants of hemagglutinin have been found in different strains of flu...H1, H2 and H3 have been involved in pandemics while H5, H7 and H9 variants have also been found in humans)

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Lysis (6)

- The cell lyses (bursts), releasing lots of new bacteriophages that can then infect new cells

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Penetration (2)

- The genome of the bacteriophage is injected into the bacteria (NOTE: Because the capsid of the virus never enters the bacteria, there is NO uncoating stage)

What does the enzyme neuraminidase do?

- This enzyme chews up the glycan molecules on the envelope of the newly emerged flu particles (NOTE: This keeps the new viral particles from sticking to each other, so they can go find new, uninfected host cells!) (EXTRA: At least nine variants of neuraminidase have been identified, but typically only N1 and N2 variants are found in humans)

The Life Cycle of a Bacteriophage (or, Lytic Cycle): Maturation (5)

- Viral enzymes chew up the bacteria from the inside out

What are bacteriophages?

- Viruses that infect ONLY bacteria

How are microbiologists able to study viruses?

- We can see them with an electron microscope and we can grow them in culture (as long as we have the right cells) - We can grow the cells, infect them with viruses and then harvest the viruses once the cells have made a bunch of them (NOTE: We can grow LOTS of cells, including: liver cells, kidney cells, muscle cells, heart cells, neurons, intestinal cells...)

How do we know when the cells being studied are infected and have produced viral particles?

- When the cells lyse, they leave empty spaces called plaques where cells should have been, but aren't because they have lysed

Can "H" and "N" variants be mixed and matched?

- YES! "H" and "N" variants can be mixed and matched in different host cells, producing different strains of flu (NOTE: H1N1 - caused the 1918 pandemic & returned in 2009; H2N2 - killed around 1.5 million people in 1957; H3N2 - the most prevalent and troublesome form of Influenza A since 1968)

Why study bacteriophages in a CLINICAL microbiology class?

1. As the natural predators of bacteria, bacteriophages have been proposed as alternatives to antibiotics for many antibiotic resistant bacterial strains (i.e., species-specific biological antimicrobials) (THINK: A bacteriophage specific S. aureus could infect the S. aureus cells, multiply inside of the cells, and then kill them (w/o killing a lot of beneficial bacteria in the process!)) 2. Sometimes bacteriophages can transfer new traits to bacteria through a process called lysogenic conversion (NOTE: Sometimes phage genes inserted into bacterial genomes can cause the production of toxins or pathological enzymes, creating really nasty pathogens like Vibrio cholerae, Clostridium botulinum, and Corynebacterium diphtheriae)

Flu Vaccine: Complicated to Create

1. The flu keeps changing! (NOTE: the flu can be one of a number of variants, so scientists try to predict which variants will be most prevalent in a given season) 2. The flu virus has up to eight RNA strands in its nucleocapsid. So, sometimes those animals susceptible to flu infection are infected with MORE than one influenza type! (NOTE: Reassortment can lead to completely new strains of influenza with new associated symptoms)

Compare and Contrast: Bacteriophage v. Animal Virus Multiplication

Refer to Table 6.5 (Nunez PPT 3, Slide 10)