Order 755765: One shot virus annual influenza vaccination

Influenza

Part 1

Aims of the lecture

To provide an introduction to the influenza virus and how it causes disease in humans.

Learning outcomes

• Describe the basic structure of the influenza virus

• Explain how the virus infects and replicates • Explain the importance of Haemagglutinin and Neuraminidase in viral pathogenicity

• Compare and contrast the different diagnostic techniques used to identify flu

What is influenza? • An infectious disease commonly called flu • Caused by a family of influenza viruses; A, B and C • Can infect birds and mammals including humans • Symptoms of the disease include chills, fever, runny nose, sore throat, muscle pains, fatigue, and headache

• Spread in humans by direct contact with infected individual or contaminated object and by inhalation of virus particles

• Distinct from the “common” cold, parainfluenza and stomach flu. Also different from haemophilus influenzae which is a bacterial form of respiratory disease

Influenza virus • Negative sense single stranded RNA virus – i.e cannot be directly translated to protein, must first be transcribed to sense RNA

• Influenza virus A, B and C distinguished by the nucleoprotein and the matrix protein

• All infect humans and – A infects other mammals and birds (pandemic) – B infects seals – C infects pigs and dogs

• Influenza C tends to cause mild disease

Influenza A

Influenza proteins (and nucleic acids)

Protein/Nucleic acid Function

Neuraminidase (NA) Membrane glycoprotein, cleaves sialic acid groups from host glycoproteins. Required for release from host cell

Haemagglutinin (HA) Membrane glycoprotein, major viral antigen. Required for entry and release from host cell

Matrix protein (M1) Provides strength and rigidity

M2 Ion channel required for pH maintenance

B1, PB2, PA RNA polymerases required for transcription of viral RNA

8 RNA segments Code 12 viral proteins

Influenza A • Generally thought to be the most pathogenic • Causes epidemics and pandemics (4 in the last 100 years)

• Classified according to their HA and NA subtypes – H1 – H18 (H18 found in 2013 in a Peruvian bat) – N1 – N10

• All subtypes can infect birds except H17N10 found only in bats

• Only H1N1 and H3N2 currently in circulation among humans

Antigenic variation

• Poor proof reading rates in viral polymerases leads to point mutation in viral HA (genetic drift)

• Occasionally mutated HA has an advantage and new HA strains emerge and infect

• Reassortment of HA in animals infected with more than one strain of influenza A also lead to new HA often novel to the human host

Influenza virus

Haemagglutinin • HA named because it causes haemagglutination in RBC

• Binds to sialic acid on epithelial cells to initiated viral entry

• Sialic acid is linked to galactose in different configurations

a2-3 linkage

Haemagglutinin linkages

• Isolates from different species vary in ability to bind to sialic acid/galactose – Human and swine isolates prefer Sia-Gal in an α2- 6 linkage

– Avian and equine isolates prefer α2-3 linkage

Haemagglutinin linkages • Isolates from different species vary in ability to bind to sialic acid/galactose – Human and swine isolates prefer Sia-Gal in an α2-6 linkage

– Avian and equine isolates prefer α2-3 linkage • Human epithelial cells (main target for flu) have the α2-6 configuration whereas goblet cells lower in the respiratory tract have α2-3

• Swine respiratory tract has both binding sites (and possibly more variation)

• Pigs act as mixing bowl

Neuraminidase

• HA binds to sialic acid on cell so prevents release of new virion after budding

• NA cleaves sialic acid from sugar allowing virion to be released

• Cleavage is not equivalent by all subtypes • Most strains have a preference for the α2-3 configuration

Viral replication

1. Virion is endocytosed

2. RNA released into the cytosol and then enter the nucleus 3b. Transcription occurs

6. NEP and M1 traffic newly replicated RNA to the membrane

3a-4. Transcribed RNA is transported to cytoplasm for translation or remains in nucleus

5. New viral proteins are synthesised. 5a. Other proteins returned to nucleus to form new vRNA particles 5b. NA and HA transported to membrane.

7. New virions bud off

Influenza • Occurs seasonally–generally between October and April in

the UK • Symptoms include:

– High temperature – Aches and pains in joints – Headaches – Watery eyes – Fatigue – Sore throat and watery discharge from nose – Diarrhoea

• At risk groups given flu vaccine seasonally • Nasal spray vaccine for 2, 3 and 4 year olds introduced in

September 2014

Why is flu seasonal? • People are indoors leading to increase in transmission • Less ultraviolet radiation may reduce the likelihood of the virus being

damaged or killed • Cold temperatures lead to drier air, which may dehydrate mucus

membranes • Aerosol transmission of the virus is enhanced when the air is cold and

dry – Virus degraded in moist air – Infected hosts shedding the virus for a longer period of time in cold

• The virus may linger longer on exposed surfaces (doorknobs, countertops, etc.) in colder temperatures

• Influenza virus has a "butter-like coating". The coating melts when it enters the respiratory tract – Cold - coating becomes a hardened shell – Warm - coating melts before the virus reaches the respiratory tract

Transmission of flu • Inhaling infected droplets when people sneeze, cough or speak – Indirect – Up to 1 metre

• Inhaling droplets through direct contact – Kissing

• Indirect transmission (fomite transmission) – e.g. hand to mucous

Diagnosing flu

H1N1 09

• Spread to 214 countries • 18,306 deaths • High rates in infants and children • Leads to other complications • Early diagnosis of disease and strain can help to prevent transmission

Clinical presentation Generally if you are fit an healthy there is no need to be formally diagnosed with flu and your immune system will fight the infection Signs and symptoms Influenza Cold Symptom onset Abrupt Gradual Fever Usual; lasts 3-4 days Rare Aches Usual; often severe Slight Chills Fairly common Uncommon Fatigue, weakness Usual Sometimes Sneezing Sometimes Common Stuffy nose Sometimes Common Sore throat Sometimes Common

Chest discomfort, cough Common; can be severe Mild to moderate; hacking cough

Headache Common Rare

Laboratory diagnostics • Virus culture – Nasopharyngeal swabs or aspirates – Inoculated onto a cell culture monolayer (Human Fibroblast, Rhesus Monkey Kidney)

– Monitored by light microscopy – Sensitive • Antibody testing – Blood samples – Haemagglutination inhibition assay (HIA) – Enzyme immunoassay (including most rapid tests) 50- 70% sensitivity, 90-95% specificity

• PCR

Viral Haemagglutination assay • HA on flu virus causes RBC to form a lattice – called haemagglutination

• Dilutions of virus mixed with the same quantity of RBC added to a microtitre plate

• Look for when RBC start to show haemaggluntination – viral titre

Haemagglutination inhibition assay

• Carry out haemagglutination assay and determine the HA viral titre

• Add virus to microtitre plate • Add serial dilutions of serum samples to be tested

• Add RBC and incubate • What happens?

The microtitre plate

Virus

Serum

RBC

Red blood cells and flu virus together cause haemagglutination Serum from patients with flu will have antibody against the virusWhen you add red blood cells they are inhibited from binding to the virus

• Any serum samples containing antibodies to influenza will bind to the virus and prevent it sticking to the RBC. Haemagglutination is therefore inhibited.

• Antibodies develop very quickly after flu infection

• Symptoms and presence of antibodies confirm disease

• Antibodies found after disease will confer some protection

Summary

• Influenza caused by influenza virus A, B or C • Influenza A most pathogenic – Antigenic variation – High mutation rate

• HA and NA important for infection and spread of virus

• Highly contagious • Most people can clear the virus • Several tests to diagnose the disease