Viruses Chapter 5, 25 & 37

Viruses

Major cause of disease– also importance as a new source of

therapy– new viruses are emerging

Important members of aquatic world– move organic matter from particulate

to dissolved Important in evolution

– transfer genes between bacteria, others

Important model systems in molecular biology

Viral replication

Video

General Properties of Viruses

Virus: genetic element that cannot replicate independently of a living (host) cell

Virus particle (virion): extracellular form of a virus, complete virus particle.

– Exists outside host and facilitates transmission from one host cell to another

– Contains nucleic acid genome surrounded by a protein coat and, in some cases, other layers of material

Viral Hosts and Taxonomy

– Viruses can be classified on the basis of the hosts they infect

Bacterial viruses (bacteriophages)Archaeal virusesAnimal virusesPlant viruses

Viruses come in many shapes and sizes– Most viruses are smaller than prokaryotic cells;

range from 0.02 to 0.3 µm

Most viral genomes are smaller than those of cells

Nature of the Virion

Virion size range is ~10–400 nm in diameter and most viruses must be viewed with an electron microscope

– Capsid: the protein shell that surrounds the genome of a virus particle.Composed of a number of protein molecules arranged in a precise and highly repetitive pattern around the nucleic acid

– Nucleocapsid: complete complex of nucleic acid and protein packaged in the virion.

– Capsid can be more or less complex and composed of only one or more than one type of proteins.

– Capsomere: subunit of the capsidSmallest morphological unit visible with an

electron microscope

Helical Capsid Helical capsid are shaped like hollow tubes with

protein walls (Tobacco Mosaic Virus is the best studied example)

Icosahedral Capsids An icosahedron is a regular polyhedron with 20

equilateral faces and 12 vertices Capsomers

– ring or knob-shaped units made of 5 or 6 protomers– pentamers (pentons) – 5 subunit capsomers– hexamers (hexons) – 6 subunit capsomers

Capsids of Complex Symmetry

Some viruses do not fit into the category of having helical or icosahedral capsids– poxviruses – largest animal virus– large bacteriophages – binal symmetry

head resembles icosahedral, tail is helical

Bacteriophage

Bacteriophage are viruses that infect bacteria.

Nature of the Virion– Enveloped virus: virus that contains additional layers

around the nucleocapsid– Animal virus envelopes (lipids and carbohydrates) usually

arise from host cell plasma or nuclear membranes

Nature of virion

Some virions contain enzymes critical to infection

– Lysozyme Makes hole in cell wallLyses bacterial cell

– Nucleic acid polymerases– Neuraminidases

Enzymes that cleave glycosidic bonds

Allows liberation of viruses from cell

Morphology of different viruses

Viral genome

Diverse nature of genomes A virus may have single or double stranded

DNA or RNA The length of the nucleic acid also varies

from virus to virus Genomes can be segmented or circular

Viral replication

Viral Attachment and Penetration

Bacteriophage T4: virus of E. coli; one of the most complex penetration mechanisms

– Virions attach to cells via tail fibers that interact with polysaccharides on E. coli cell envelope

– Tail fibers retract and tail core makes contact with E. coli cell wall

– Lysozyme-like enzyme forms small pore in peptidoglycan

– Tail sheath contracts and viral DNA passes into cytoplasm

Attachment of bacteriophage T4 to the cell wall of Escherichia coli and injection of DNA

Viral Entry and Uncoating

Assembly

Virion Release

Isolation, cultivation, identification

Bacteriophages can be grown

1- In suspension of bacteria in liquid media

2- In bacterial cultures on solid media.On solid media the phage infection

produces plaques that can be counted and theoretically correspond to one virus per plaque. The count is given as pfu (plaque forming units).

Quantification of bacterial virus by plaque assay using the agar overlay technique

Isolation, cultivation, identification

Animal viruses can be grown in– Living animals (mice, rabbits, and

guinea pigs)– Embryonated eggs – Cell cultures

Identification can be difficult. Most common are serological methods. Molecular methods such as PCR are becoming routine for the identification of some viruses.

Cell cultures in monolayers grown on a Petri plate

Quantification of Animal Viruses Intact Animal Methods

– Some viruses do not show recognizable changes in cell cultures yet cause death or disease in whole animals

– Virus is diluted– Animals are infected with viral dilution– End point is calculated (LD50 or ID50)

Mechanisms to diminish viral infectionsEukaryotes possess mechanisms to diminish viral

immune defense mechanismsProkaryotes also possess mechanisms

– Restriction modification system– DNA destruction system; only effective against

double-stranded DNA viruses– Restriction enzymes (restriction endonucleases)

cleave DNA at specific sequences– Modification of host’s own DNA at restriction enzyme

recognition sites prevents cleavage of own DNAViral mechanisms to evade bacterial restriction systems

– Chemical modification of viral DNA (glycosylation or methylation)

– Production of proteins that inhibit host cell restriction system

Classification of virusesClass Type of

nucleic acidBacterial Animal

Class I dsDNA Lambda, T4 Herpes

Class II ssDNA ФX 174 Chicken anemia

Class III dsRNA Ф6 Reoviruses

Class IV ssRNA (+) MS2 Polio

Class V ssRNA (-) Influenza

Class VI ssRNA. DNA intermediate

Retroviruses

Class VII dsDNA. RNA intermediate

Hepatitis B

Viral replication

Class I, the DNA has the same structure as cellular DNA and can be used to produce mRNA and copies of the viral genome.

Class II, the ss DNA must be copied into its complementary strand that can be used for transcription or as template for making more copies of the genome.

Class III, viruses require the presence of a viral enzyme that copies dsRNA into mRNA and that makes copies of the genome

Viral replication

Class IV viruses can use the RNA directly as mRNA to synthesize viral proteins.

Class V viruses require a viral enzyme to transform the negative strand RNA into mRNA.

Class VI viruses need reverse transcriptase to transcribe the RNA into DNA that is then used to produce mRNA and copies of the genome.

Production of Viral Protein– Production follows synthesis of viral mRNA

Early proteins – synthesized soon after infection– necessary for replication of virus nucleic

acid– typically act catalytically – synthesized in smaller amounts

Late proteinsSynthesized later Include proteins of virus coatTypically structural componentsSynthesized in larger amounts

Overview of Bacterial Viruses

Bacteriophages are very diverse Best-studied bacteriophages infect enteric

bacteria– Examples of hosts: E. coli, Salmonella enterica

Most phages contain dsDNA genomes Most are naked, but some possess lipid

envelopes They are structurally complex, containing

heads, tails, and other components

Schematic representations of the main types of bacterial viruses

Overview of Bacterial Viruses

Viral Life Cycles– Virulent mode: viruses lyse host

cells after infection– Temperate mode: viruses

replicate their genomes in tandem with host genome and without killing host

Virus can also be lytic

Temperate Bacteriophages, Lambda, and P1

Temperate viruses: can undergo a stable genetic relationship within the host

– But can also kill cells through lytic cycleLysogeny: state where most virus genes are

not expressed and virus genome (prophage) is replicated in synchrony with host chromosome

Lysogen: a bacterium containing a prophageUnder certain conditions lysogenic viruses

may revert to the lytic pathway and begin to produce virions

Lytic and lysogenic cycles

Lytic and lysogenic

Temperate Please click on the link to watch this video

Viral infection

When bacterial viruses are lysogenic the virus that is integrated in the bacterial genome is called a prophage.

When animal and plant viruses are lysogenic the integrated virus is called a provirus.

The viral genome is replicated together with the host cell genome and is passed on to the daughter cells. Environmental conditions can influence the life cycle and stimulate a lysogenic virus to become lytic (UV irradiation, X rays)

Overview of Animal Viruses

Consequences of Virus Infection in Animal Cells

– Persistent infections: release of virions from host cell does not result in cell lysisInfected cell remains alive and

continues to produce virus – Latent infections: delay between

infection by the virus and lytic events– Transformation: conversion of normal

cell into tumor cell– Cell fusion: two or more cells become

one cell with many nuclei

Possible effects that animal viruses may have on cells they infect

Retroviruses

Genome: ssRNA (two copies in each virion) Complex virus: it carries enzymes in the

virion that are essential for replication (reverse transcriptase)

Enveloped virus. On the envelope there are proteins that can bind to the receptor on the host cell.

All retroviruses have their genome organized as follows: gag – pol – env. Gag: structural proteins, pol: reverse transcriptase and integrase, env: envelope proteins.

Retroviruses

Retroviruses have a unique genome– Two identical ssRNA molecules of the

plus (+) orientation– Complex virus: it carries enzymes in the

virion that are essential for replication (reverse transcriptase)

– Enveloped virus. On the envelope there are proteins that can bind to the receptor on the host cell.

Retroviruses

Retroviruses have a unique genome

– Contain specific genesgag: encode structural proteinspol: encode reverse transcriptase

and integraseenv: encode envelope proteins

Retrovirus structure and function

HIV life cycle

Virion has viral protein spike, gp120 – attaches to CD4 cells

(T helper cells and other cells) and co-receptors CCR5 and CXCR-4

Integrates into host cell’s DNA as a provirus

Can remain latent – asymptomatic

HIV life cycle

CDC Classification System for Stages of HIV-Related Conditions

Acute– 2–8 weeks after infection– most experience brief illness called acute

retroviral syndrome– rapid multiplication and dissemination of

virus throughout body– stimulation of immune response

Asymptomatic (latent)– may last from 6 months to 10 or more

years– levels of detectable HIV in blood decrease,

although viral replication continues– effects on immune functions may occur

CDC Classification System for Stages of HIV-Related Conditions

Chronic symptomatic– formerly called AIDS-related complex – can last for months to years– viral replication continues– numbers of CD4+ cells in blood significantly decrease

results in patients developing a variety of illnesses often caused by opportunistic pathogens and AIDS related cancers

AIDS – fourth and last stage– immune system no longer able to defend against virus

Definition of AIDS– all HIV-infected individuals who have fewer than 200

CD4+ T cells/microliter of blood or a CD4+ cell percentage of lymphocytes of less than 14

Diseases associated with AIDS

Animal ssRNA (+)

Poliovirus and coronavirus.Positive strand means that the genome

is also the mRNA and can be used immediately to make viral proteins.

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.

Poliovirus

Poliovirus

Polio is slowly becoming a reemerging disease. At one point in history polio was almost under total control because of the presence of an effective vaccine.

The virus encodes all the proteins as a single giant protein (2200 aminoacids) called a polyprotein that is later cut into about 20 different proteins. The virus also encodes for the protease that can cleave the polyprotein.

Replication happens in the cell cytoplasm. Nonenveloped

Positive-Strand RNA Animal Viruses

Highly contagious viral disease caused by the SARS-associated corona virus (SARS-CoV) – transmitted by droplet spread; can be fatal– Sudden onset, severe illness in healthy individuals– dry cough develops in days; most will develop

pneumonia No specific treatment is currently approved

Coronavirus

Animal ssRNA (-)

The viral genome is negative strand and needs to be transformed into positive strand through RNA-dependent RNA polymerase.

The genome can be transcribed as small mRNAs or as the whole genome.

Examples of negative strand RNA viruses are rabies and influenza virus.

In the case of influenza the genome is segmented and is present in the virion as 8 separate pieces. Replication of the genome happens in the nucleus.

Replication of a negative-strand RNA virus

Influenza virus

Replication cycle of Influenza A virus

Antigenic shift

Animal ssRNA (-)

Another mechanism used by these viruses to avoid the immune system is antigenic drift.

In antigenic drift the surface proteins of the virus are altered by mutations that occur in the genome of the virus.

Influenza can be prevented with vaccine prophylaxis that must be repeated every year and that is strongly suggested for individuals that risk death from infection with the flu virus.

Rabies is treated with postexposure prophylaxis (vaccine and immunoglobulins are injected after exposure).

Influenza (Flu)

An important feature of the influenza viruses is the frequency with which changes in antigenicity occur– Antigenic drift – due to accumulation

of mutations in a strain within a geographic area

– Antigenic shift – due to reassortment of genomes when two different strains of flu viruses (from humans and animals) infect the same cell and are incorporated into a single new capsid

Antigenic drift

Ebola Hemorrhagic Fever

Ebola-Member of single-stranded, negative sense RNA family

Infection is severe and ~80% fatal No known carrier state; fruit bat may be

reservoir Transmission from direct contact with Ebola

victim, body fluids or clinical samples Internal hemorrhaging Supportive therapy; no

treatment available Experimental vaccines

being evaluated

dsDNA virus - Herpesvirus

HSV (herpes simplex virus, varicella zoster virus, CMV (cytomegalovirus) Epstein-Barr virus.

dsDNA virus can integrate into the genome of the host and this gives them the ability to remain latent.

HSV and Varicella zoster remain latent in the neurons of the sensory ganglia from which they can reemerge periodically.

Viral DNA is uncoated in the nucleus. Assembly of the virus is on the nuclear

membrane and envelope is of nuclear origin.

Herpesvirus

dsDNA virus - Herpesvirus HSV-1 belongs to herpesviruses.

– Herpes simplex virus 1 (HSV-1) is transmitted by oral or respiratory routes and produces cold sores. It remains latent in the trigeminal nerve ganglia. Recurrence can be triggered by events such as stress, UV exposure, emotional upsets, and hormonal changes.

HSV-1 Cold sores

Genital Herpes Caused by HSV-2 Clinical manifestations

– fever, burning sensation, genital soreness, and blisters in infected area

– blisters heal spontaneously, but virus remains latent and is periodically reactivated

Can be treated with antiviral drugs (e.g., acyclovir) Congenital (neonatal) herpes

– usually HSV-2– one of the most life-threatening of all infections

in newborns– ~1,500–2,200 babies/year in U.S.– can result in neurologic involvement and

blindness– Caesarian section recommended

dsDNA virus - Herpesvirus

Chickenpox

Varicella zoster virus

Chickenpox (Varicella) and Shingles (Herpes Zoster)

Shingles (herpes zoster; postherpetic neuralgia)– reactivated form of

chickenpoxvirus resides in

cranial and sensory neurons

reactivation - virus migrates down neuron

Treatment – supportive; acyclovir

and others

Chickenpox (Varicella) and Shingles (Herpes Zoster)

dsDNA virus - Herpesvirus

Herpesviruses can also be tumorigenic. EBV can cause Burkitt’s lymphoma that is the most

common cause of childhood cancer in Africa. EBV can cause also nasopharyngeal carcinoma

(Southeast Asia). EBV is also the cause of a very mild disease called

mononucleosis. CMV is also very widely spread and the cause of

mild disease. All these viruses can cause serious disease in

immunocompromised patients.

Epstein Barr virus (EBV)

Mononucleosis (Infectious) Caused by Epstein-Barr virus

(EBV)– herpes virus, dsDNA,

icosahedral with envelope– infects B cells– also associated with Burkitt’s

lymphoma and nasopharyngeal carcinoma

Spread by mouth-to-mouth contact

Clinical manifestations– enlarged lymph nodes and

spleen, sore throat, headache, nausea, general weakness and tiredness, and mild fever

– self-limited disease, lasting 1 to 6 weeks

Burkitt's Lymphoma

dsDNA – Pox viruses

Pox viruses replicate in the cytoplasm. Viral DNA synthesis occurs outside the

nucleus. They do not have an envelope, but are

covered on the surface with protein tubules arranged in a membrane-like pattern.

Smallpox was caused by this virus but the disease has been officially declared eradicated after effective vaccination of the human population.

During the Middle Ages and estimated 80% of the Europeans contracted smallpox. Those who recover had disfiguring scars.

Smallpox (Variola) Caused by variola virus

– large, brick-shaped complex virus– linear dsDNA

Transmitted by aerosol or contact– humans are the only natural host

Clinical Forms of Smallpox Variola major

– most common, severe form – extensive rash and higher fever– 33% fatality rate

Variola minor– less common form and less severe– fatality rates of 1% or less

Both forms usually transmitted by direct and fairly prolonged face-to-face contact

Smallpox (Variola)

Eradication of Smallpox 1977 – last case from a natural infection

occurred in Somalia Why eradication was possible –

– disease has obvious clinical features– humans are only hosts and reservoirs – there are no asymptomatic carriers– short infectivity period (3–4 weeks)

dsDNA viruses

The vaccinia virus is a close relative of pox viruses and has been very important for the development of vaccines.

Adenoviridae cause the common cold. Papillomavirus causes warts, but some

strains are capable of causing cancer Hepdnaviruse causes Hepatitis B virus all

other forms of hepatitis are caused by RNA viruses

Hepatitis

– inflammation of liver, caused by 11 different viruses2 herpesviruses—Epstein-Barr virus (EBV) and

cytomegalovirus (CMV)– cause mild, self-resolving disease– no permanent hepatic damage– signs and symptoms include fatigue, nausea, and

malaise

Hepatitis A & E viruses (HAV) & (HEV)

HAV- icosahedral, naked virus,positive strand linear RNA

Spread by fecal-oral contamination of food, drink, or shellfish

Clinical manifestations– usually mild intestinal infection

anorexia, general malaise, nausea, diarrhea, fever, and chills

– occasionally viremia occurs leading to liver infection jaundice

HEV-implicated in many epidemics in developing countries

transmission by fecal contaminated water similar to HAV course of disease ~15%–25% fatality rates in pregnant women

Hepatitis B virus (HBV)

– dsDNA virus– Dane particle is

infectious virion– transmitted through

body fluids and intra-

venous equipment– can pass the placenta

and breast milk– ~1.25 million chronically

infected in U.S., 200 million worldwide

Hepatitis C& D Virus (HCV) & (HDV)

HCV-RNA virus Transmission virus contaminated blood, fecal

oral route, also spread from mother to fetus, and through organ transplants

Epidemic with more than 1 million new cases/yr in U.S.

HDV -Causes severe acute and chronic hepatitis in HBV infected

Treatment, prevention, and control– serological tests for anti-HDV antibodies– no satisfactory treatment

dsRNA viruses - rotavirus Non enveloped and cause of diarrhea in infants. Replication takes place in the cytoplasm. Genome is segmented. The RNA replication is guaranteed by the

presence in the virion of a RNA-dependent RNA polymerase that can copy RNA from RNA.

Released by cell lysis.

Viruses and Cancer

Complex, multistep process Often involves oncogenes

– cancer causing genes– may come from the virus OR may be transformed

host proto-oncogenes (involved in normal regulation of cell growth/differentiation)

Viroids and virusoids

Viroids are infectious agents that consist of only RNA. Cause over 20 different plant diseases.

They are covalently closed circular ssRNA about 250-370 nucleotides long.

The RNA of viroids does not codify for any viral product, but it is replicated by the host RNA polymerase. Probably because of its structural properties the cell enzyme uses the viroid as template to make copies.

Viroids and virusoids

Virusoids are similar in structure to viroids, but they codify for one or more gene products and they need a helper virus to be able to infect a cell.

The best studied virusoid is the human hepatitis D virus that uses hepatitis B as helper virus.

If a host cell contains both hepatitis D and B viruses, the virusoid RNA and its gene product (delta antigen) become able to infect other cells and replicate.

Prion Prions (Proteinaceous

infectious particles) cause neurodegenerative disease (transmissible spongiform encephalopathies).

(PrPC) encodes for the normal form of prion

(PrPSc ) encodes for the abnormal form of a cellular protein.

Entrance of PrPSc induces a conformational change in the normal protein and transforms it into the abnormal form

Prions – Proteinaceous Infectious Particle

Cause a variety of degenerative diseases in humans and animals– scrapie in sheep– bovine spongiform encephalopathy (BSE) or

mad cow disease– Creutzfeldt-Jakob disease (CJD) and variant

CJD (vCJD) in humans– kuru in humans

All prion caused diseases– have no effective treatment– result in progressive degeneration of the brain

and eventual death

Prion overview

video

Prion characteristics

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