MBCHB III

Mrs L Charters

Virology 2011

Lecture Series Outline

This series of lectures aims to achieve two main objectives:

1. To give students a basic understanding of viral replication processes and how they may relate to the development of vaccines and anti-viral drugs

2. To provide students with a specific understanding of the pathophysiology, epidemiology and replication strategies of:

– Rabies virus

– Influenza virus

– Herpes simplex virus

Introduction

To understand viral structure, replication and the mechanisms of virus-mediated disease you must have a thorough understanding of basic cell biology

We are going to review the following:

• Mammalian cell structure

• Transcription

• Translation

• Basic steps of viral replication

Mammalian Cell Structure

Schematic of Typical Animal Cell, Showing Subcellular Components

(1) Nucleolus (2) Nucleus (3) Ribosome (4) Vesicle (5) Rough ER (6) Golgi Apparatus (7) Cytoskeleton (8) Smooth ER (9) Mitochondria (10) Vacuole (11) Cytoplasm (12) Lysosome

(13) Centrioles

The Nucleus

• The nucleus houses the DNA genome of eukaryotic cells

• The nuclear membrane ensures the genome is protected from cellular enzymes and metabolic processes

• Transcription of genes into mRNA, tRNA and ribosomes occurs in the nucleus

• Lengths of DNA, encoding genes, are arranged around protein molecules, such as Histones, to form chromosomes

• Nuclear envelope: a double membrane that contains pores (± 9nm wide) which allow the passage of small molecules and ions, the entry of specific targeted proteins and the exit of RNA molecules

Endoplasmic Reticulum

• Interconnected network of tubules, vesicles and cisternae

• Smooth ER synthesizes lipids and steroids, metabolizes carbohydrates and steroids, attachment of receptors on cell membrane proteins, regulates calcium levels and drug detoxification.

• Rough ER is studded with ribosomes in the process of translating proteins for the secretory pathway

• Proteins destined for secretion or integration into the membranes of the “Cellular Secretory Pathway” are synthesized by ribosomes on the Rough ER membrane

• Rough ER forms a part of the secretory pathway and plays a key role in transporting integral membrane proteins destined for cell membrane and proteins destined for cellular secretion

• Vesicles containing the synthesized proteins bud off from the Rough ER and fuse with the Golgi membranes, releasing their proteins into the Golgi lumen

Golgi Apparatus

• The Golgi processes and packages macromolecules such as lipids and proteins for secretion or use within the cell

• Key processes include glycolysation and phosphorylation of proteins

• Comprises stacks of membranous cisternae with a lumen where molecular processing takes place

• Vesicles from the RER fuse with the Cis Golgi surface and progress through the stack to the Trans Golgi surface where they are targeted to their final destination such as the cell membrane

• Cells that secrete large amounts of protein molecules, e.g. white blood cells secreting antibodies, tend to have larger Golgi bodies

Transcription

• Transcription is the process of generating a complimentary RNA molecule from a sequence of DNA

• Transcription involves an RNA polymerase enzyme that binds the promoter sequence on a gene and then catalyses the synthesis of an RNA molecule from the DNA template

• Nascent RNA molecules are processed (rRNA into ribosomal subunits and tRNA into folded molecules) and transported out of the nucleus into the cytoplasm for translation

Translation

– Messenger RNA (mRNA) encodes the amino acid sequence

– Transfer RNA (tRNA) recruits amino acids in the cytoplasm

• Translation is the process of generating a polypeptide chain from an mRNA template

• Translation is mediated by three types of RNA molecules:

– Ribosomal RNA (rRNA) makes up the ribosomal subunits that mediate translation

Classification of Viruses

Baltimore Classification

• First defined in 1971

• Places viruses in 7 groups based on:

1. Type of nucleic acid (DNA or RNA) 2. Strandedness (single or double) 3. Sense (positive or negative)4. Method of viral mRNA synthesis

• Often a more reliable system as classification according to genome means viruses in a group will replicate in a similar manner

• Classification according to morphology and/ or disease is problematic as unrelated viruses can either look similar or cause similar infections

Baltimore Classification

Rhabdoviridae

Focus on Rabies Virus

Class V

Class V viruses have -ve sense ssRNA genomes and include:

Virus Family Capsid GenomeInfluenza viruses A, B & C Orthomyxoviridae Enveloped, helical -ve ssRNA

Measles virus Paramyxoviridae Enveloped, helical -ve ssRNA

Mumps virus Paramyxoviridae Enveloped, helical -ve ssRNA

Rabies virus Rhabdoviridae Enveloped, cylindrical -ve ssRNA

Ebola virus Filoviridae Enveloped, helical -ve ssRNA

Marburg virus Filoviridae Enveloped, helical -ve ssRNA

Rabies Virus

• Class: V

• Order: Mononegavirales

• Family: Rhabdoviridae

• Genus: Lyssavirus

• Species: Rabies virus

Classification:

Structure

• Helical symmetry producing a cylindrical morphology (“bullet-shaped”)

• 180 nm x 75 nm in size

• ss –ve RNA genome is tightly bound by the ribonucleoprotein core

• Matrix proteins packed between the envelope and the core

• Glycoprotein G spikes protrude from the envelope

Rabies Virus

1. Attachment and cell entry

2. Uncoating

3. Viral genome transcription

4. Viral protein translation

5. Viral genome replication

6. Progeny virion assembly

7. Progeny virion release

Virus Replication

Viruses generally display 7 distinct steps in their replication cycle:

Rabies Virus Replication

• Transcription and RNA replication occur in the cytoplasm inside Negri bodies that essentially act as “virus factories”

• Negri bodies are 2 – 10 μm in size and are specific for Rabies virus replication and are used as a definitive diagnostic histological feature

Rabies Virus Replication

Rabies Virus Replication

• Glycoprotein G spikes attach to specific receptors on the host cell surface

• The virion is taken into the cell by receptor-mediated endocytosis

1. Attachment and Cell Entry

• The low pH of the endosome induces the fusion of the viral envelop with the endosome membrane, releasing the virus genome into the cytoplasm

2. Uncoating

Rabies Virus Replication

• The viral P protein associates with the viral L protein (RNA polymerase). Together the P-L polymerase transcribe the five highly conserved genes on the viral genome producing mRNAs

• The five genes encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and viral RNA polymerase (L)

3. Transcription

• The viral mRNAs are translated by host cell machinery to produce the five viral proteins

• The G protein is glycosylated in the Golgi and is carried to the cell surface by the host cell secretory pathway

4. Translation

Rabies Virus Replication

• Later in replication the M protein regulates the switch whereby the P-L polymerase begins to synthesize whole length +ve sense RNAs which are used as a template for nascent –ve ssRNA genomes

5. Genome Replication

• The N protein is packaged with the nascent viral RNAs to form ribonucleoprotein

• M protein associates with the ribonucleoprotein to form the virion matrix

6. Progeny Virion Assembly

• Nascent virions bud off from the cell membrane containing protein G

7. Virion Release

Rabies Virus Replication

Epidemiology

• Rabies occurs in 150 countries and territories

• Globally, 99% of rabies infections in humans are transmitted through dog bites

• Vaccination programs effectively control the rabies reservoir in domestic pets such as dogs, cats and rodents

• In countries such as the UK, Taiwan, Mauritius and Japan rabies has been eliminated in land-based animal reservoirs. However concern exists for airborne reservoirs such as bats

• New Zealand and Australia have never had endemic rabies infection due to the geographical isolation and strict animal import control measures

• Rabies can have a significant economic impact in countries where outbreaks result in the infection of large numbers of livestock

Epidemiology

• In the USA annual death rates have fallen from ~100 to 1 – 2 cases due to vaccination campaigns and the development of effective human vaccines and immunoglobulin treatments

• Almost all human deaths from rabies today occur in Asia and Africa

• Annual estimated deaths (WHO, 2006):

– Asia: 31 000

– Africa: 24 000

• India has the highest incidence of rabies infection of any country in the world because of the large stray dog population

• Each year 15 million people receive post-exposure prophylaxis, preventing an estimated 327 000 deaths annually

Transmission

• The rabies virus is spread through the saliva of warm blooded animals

• Domestic animals (dogs, cats, ferrets), livestock, primates, jackals, rodents, raccoons and bats are all common reservoirs

• It is most often spread by the bite of an infected animal

• Human to human transmission is rare but a few cases of transmission have been recorded with organ transplant patients

Prevention

• The most effective method of preventing the spread of rabies infection is through preventative vaccination

• An outbreak of rabies infection in a particular geographical area can be controlled with a combination of measures:

– Vaccination of all people, livestock and domestic animals in the area

– Quarantine of any animals suspected to be infected

– Culling of populations of certain species known to be spreading the virus

– Control of stray dog and cat populations

• Many countries legally require pet owners to regularly vaccinate all domestic animals

Prevention

• The original Pasteur-Roux vaccine was produced from infected rabbit nerve tissue where the virus was weakened by air drying for 5 – 10 days

• In 1967 the human diploid cell vaccine was developed but proved expensive to produce

• Today two kinds of attenuated live virus vaccine are available:

– Purified chicken embryo cell rabies vaccine, attenuated live virus vaccine

– Purified Vero cell rabies vaccine, attenuated Wistar rabies virus strain

• The vaccine confers immunity in humans and dogs for 2 – 3 years

Disease Progression

• The rabies virus infects nerve cells at the wound of entry and travels quickly along peripheral nerves to the CNS where it causes acute encephalitis

• The virus spreads to other organs with a high viral load being detected in the salivary glands, thus promoting transmission to a new host

• Fatality: 2 days – 5 years after initial infection

• Largely depends on host species

• Most hosts die within weeks

• The African yellow mongoose is known to sometimes survive infection asymptomatically for several years

Disease Progression

• Early symptoms are not diagnostically specific and include fever, headache, general weakness and discomfort

• As infection progresses symptoms include acute pain, insomnia, confusion, anxiety, partial paralysis, excitation, depression, hallucinations, agitation, hypersalivation, difficulty swallowing and hydrophobia

• Death normally follows within several days of the onset of these symptoms

• The primary cause of death is most often respiratory insufficiency

Diagnosis

Before Death:

• PCR or viral culture from skin samples

• Diagnosis can be made from urine, cerebrospinal fluid and saliva samples but is not as sensitive

After Death:

• PCR or viral culture on a brain sample

Histological Diagnosis:

• The observation of Negri bodies is 100% diagnostic for rabies in cerebral samples but they are only detected in 80% of rabies-positive cases and thus this method is not a sufficient diagnostic test on its own

Infection Management• Treatment after exposure to rabies virus is known as post-

exposure prophylaxis (PEP)

• PEP is generally successful in preventing the development of rabies if administered within 10 days of infection

• PEP is given over a 14 day period and generally consists of:

– 1 dose of human rabies immunoglobulin (day 0)

– 4 doses of rabies vaccine (days 0, 3, 7 and 14)

• As much as possible, a significant portion of the treatment should be infiltrated around the bite with the remainder being given in a deep intramuscular injection at a site distant to the vaccination site

• Patients that have received previous preventative rabies injection need only receive 2 doses of rabies vaccine on days 0 and 2 after exposure

Prognosis

• All cases of rabies infection in humans were fatal until the invention of a vaccine in 1885 by Louis Pasteur and Emily Roux

• In humans the disease is almost always fatal if the victim does not receive PEP

• In 2004 Jeanna Giese was treated with the Milwaukee Protocol and became the first person to survive a rabies infection without being administered PEP. Subsequent studies indicate that this protocol has an estimated survival rate of 8%

Prognosis

• Milwaukee Protocol

– Patient are placed in an induced coma upon onset f rabies symptoms

– Administered Ketamine, Midazolam, Ribavirin and Amantadine

– After a period of isolation (31 days) hospitalization (76 days) Jeanna was released and shows no detrimental effects of the infection to date

– A revised protocol removes the use of Ribavirin

• There have been a total of 6 survivors of rabies infection that have not received PEP

END