Session 10

Question 1

What is a virus?

Answer 1

Viruses are simple structures consisting of a delivery system and a payload. The delivery system of a virus protects it against degradation in the environment and contains structures used to bind to target cells in the host. The payload of a virus contains the genome and enzymes necessary to initiate the first steps in virus replication

Question 2

Give an overview of influenza (flu)?

Answer 2

• Flu is an acute viral infection of the respiratory tract (nose, mouth, throat, bronchial tubes and lungs)
• It is a highly infectious illness which spreads rapidly in closed communities
• Even people with mild or no symptoms can infect others
• Most cases in the UK occur during an 8 to 10 week period during the winter

Question 3

Describe the structure of the influenza virus

Answer 3

Orthomyxoviruses are spherical, enveloped viruses containing a segmented, negative strand RNA genome
Genetic material:
• (-) ssRNA – 8 genes
• encoding 11 proteins
• include 3 RNA polymerases (high error rates)

Two surface antigens:
• Haemagglutinin (H) – 18 types – binds to cells of the infected person
• Neuraminidase (N) – 11 types - releases the virus from the host cell surface

Question 4

What are the modes of transmission for the influenza virus?

Answer 4

Influenza viruses are transmitted from person to person via the respiratory route (coughing, sneezing, inhaling)

Three potential modes of transmission:
1)small-particle aerosols (<10 μm mass diameter) - remain suspended in air for many hours

2) larger particles or droplets will typically fall to the ground within 3 m of the infected person - infect individuals in direct contact.
3) viral particles could land on surfaces, where influenza viruses remain infectious - infect others through indirect contact.

Question 5

What are the barriers to entry via the respiratory route?

Answer 5

1) Respiratory epithelial cells are covered by a thick glycocalyx and tracheobronchial mucus that can trap virus particles.
Barriers to entry via the respiratory route:

2) Ciliated respiratory epithelial cells continually sweep mucus up from the lower respiratory tract into the upper respiratory tract, where it is usually swallowed.
3) In the lung, immunologic defences include secretory IgA, natural killer (NK) cells, and macrophages

Question 6

How does the influenza virus enter the cell?

Answer 6

virion with Haemagglutinin protein on the surface binds to Neu5Ac (NANA) residues – sialic acid on a glycoprotein/glycolipid which acts as a receptor for influenza virus
Entry then occurs via receptor mediated endocytosis
Virion is then released into cytoplasm.

Question 7

How does the influenza virus leave the cell?

Answer 7

Virus buds out of cell but still attached by haemagglutinin bound to glycoprotein. Neuraminidase cleaves this site releasing the virion.

Question 8

What are symptoms and complications of influenza?

Answer 8

Symptoms:
Neurological:
Fever, Headache, confusion
Respiratory:
Dry cough, Sore throat, Nasal congestion
Gastrointestinal:
Nausea, Vomiting, Diarrhoea 
Musculoskeletal:
Myalgia, Fatigue 

Complications
Neurological:
Meningitis/encephalitis
Respiratory:
Otitis media
Croup
Sinusitis/bronchitis/pharyngitis
Pneumonia

Question 9

What are the three types of influenza?

Answer 9

A, B and C.
A viruses
• cause outbreaks most years & are the usual cause of epidemics and pandemics
• live & multiply in many different animals & may spread between them
• birds, particularly wildfowl, are the main animal reservoir
B viruses
• tend to cause less severe disease (unless elderly or immunosuppressed) & smaller outbreaks
• predominantly found in humans
• burden of disease mostly in children or elderly

C virus
In humans and swine
mild disease without seasonality

Question 10

How does influenza replicate?

Answer 10

(-)ssRNA so needs to be transcribed into (+)ssRNA so it can be made into more (-)ssRNA. Simultaneously (-)ssRNA made into mRNA by RNA-dependent RNA polymerase (from infecting virus) and used to make viral proteins which can combine with the new (-)ssRNA for assembly of nucleocapsids which can leave the cell and infect other cells.

Question 11

What is the time course of influenza A virus infection?

Answer 11

• incubation period 1-5 days (average 2-3 days) • people with mild or no symptoms can still infect others
• sudden onset of fever, chills, headache, muscle/joint pain, extreme fatigue, dry cough, sore throat, stuffy nose
Recovery in 2-7 days.

Question 12

Who is at risk of complications from influenza?

Answer 12

Risk of most serious complications is higher in:
– children under six months – older people (age over 65)
– those with underlying health conditions such as respiratory disease, cardiac disease, long term neurological conditions or immunosuppression
– pregnant women (flu during pregnancy may be associated with perinatal mortality, prematurity, smaller neonatal size and lower birth weight) including up to 2 wks post partum
– Morbid obesity (BMI≥40)

Question 13

How do we diagnose flu?

Answer 13

Normally by judging symptoms
If patient is hospitalised then run tests or if higher mortality than normal in flu season.
Generally use sample from a nasopharyngeal swab for rapid test. (uses antigen detection)

Question 14

What are the treatments for influenza ?

Answer 14

1) Antivirals e.g. rimantadine and amantadine - which inhibit viral uncoating after uptake probably through M2 protein influenza A, not really given due to increasing resistance
2) Neuraminidase Inhibitors e.g. Oseltamivir (Tamiflu), Zanamivir (Relenza), - which inhibit viral release from the infected cell & cause aggregation of viral particles influenza A & B
3) Prevention
a. formalin-inactivated vaccine (quadrivalent/trivalent) by injection influenza A & B
b. live, attenuated, cold-adapted vaccine (quadrivalent) by nasal spray influenza A & B

Question 15

How do neuraminidase inhibitors work?

Answer 15

Blocks neuraminidase and blocks the virus from leaving so preventing spread and reproduction.

Question 16

Describe the genetic change in influenza A and B

Answer 16

The flu virus is constantly replicating
Life cycle of approximately 6 hours
Viral RNA polymerases have a high error rate
High error rate & lack of proofreading ability leads to mutations
Genetic variation and resistance
Genetic changes in the influenza virus

B
Surface antigens hemagglutinin & neuraminidase (antigenic drift)
This refers to minor antigenic changes in H and N proteins that occur each year. Antigenic drift does not involve a change in the viral subtype. This phenomenon can be easily explained by random mutations in viral RNA and single or a small number of amino acid substitutions in H and N proteins.

A
Dramatic changes in the antigenic properties of the H and/or N proteins
Change in subtype, e.g., from H1N1 to H3N2
Occurs infrequently - maybe every 10 or 20 years
Only influenza type A viruses show antigenic shift
Antigenic shift
Genetic changes in the influenza A virus
Surface antigens hemagglutinin & neuraminidase from different species
This refers to major changes in H and N proteins that occur because the influenza viruses from several species occur in the same host. It does involve a change in the viral subtype resulting in different H and N proteins. This phenomenon can be easily explained by the reassortment of different RNA segments from each species in a new capsid. Potentially no previous immunity to new subtype.

Question 17

What is antigenic drift?

Answer 17

antigenic drift: minor changes (natural mutations) in the genes of flu viruses that occur gradually over time – cause seasonal epidemics

Question 18

What is antigenic shift?

Answer 18

antigenic shift: major changes in the genes of flu viruses that occur suddenly when two or more different strains combine. This results in a new subtype – cause widespread epidemics/pandemics

Question 19

How does reassortment occur in antigenic shift for influenza?

Answer 19

a) influenza type A viruses in many animals, including horses, pigs, and wild migrating waterfowl. b) reassortment can occur between influenza A viruses that infect different animal and avian species, e.g., pigs can be infected by human- and avian-specific influenza viruses c) In environments where pigs, birds and humans coexist, it is possible for a pig to be simultaneously infected with multiple influenza subtypes.
d) “Reassortants” can, therefore, be produced within one host animal (the pig), in which the mRNAs encoding the H and N antigens have been reassorted into unique combinations.
e) The reassortant virus then has the potential to spread among humans, birds, and pigs

Question 20

What are the consequences of antigenic shift?

Answer 20

Antigenic shift leads to a new subtype of influenza virus
May not have been seen in circulation in population for many years Immune systems of many individuals have no defence against this new subtype
Leads to epidemic and pandemic

Question 21

What causes symptoms?

Answer 21

SYMPTOMS ≡ the body’s immune response to viral invasion Antibodies triggered & immune cells move to site of infection Release of cytokines leading to local inflammation

Question 22

How does flu actually kill people?

Answer 22

a) The immune system “overreacts” – T-cells attack and destroy the tissues in which the virus is replicating – in particular the lungs
b) There is an opportunistic secondary infection – i.e. bacterial ( Streptococcus or Staphylococcus species) - usually in the lungs

Question 23

Who is recommended for flu vaccinations

Answer 23

• pregnant women at any stage of pregnancy
• children aged between 6 months to 5 years
• elderly individuals (aged more than 65 years)
• individuals with chronic medical conditions
• health-care workers.

Question 24

How do we describe E.coli

Answer 24

Escherichia coli
• Gram-negative rods (red stain) 
• Typically lactose-fermenting 
• Facultatively anaerobic 
• Often motile 
• Numerous serotypes 
• Constituent part of large bowel microbiota of many animals, including humans

Question 25

How do we identify E coli in the laboratory?

Answer 25

* E coli and other Enterobacteriaceae can use the sugar lactose as an energy source, producing lactic acid as a waste product. * Pseudomonas aeruginosa cannot use lactose –it is a non-lactose fermenter * MacConkey agar contains lactose and a pH indicator that goes red with acid pH * E coli and other Enterobacteriaceae grow as pink colonies on MacConkey agar. Non-lactose fermenters grow as yellow colonies

Question 26

Describe the ecology of E. coli?

Answer 26

The transmission of E coli between animals , humans and the environment , including pathogenic and antibiotic resistant strains, illustrates the challenges of controlling disease and the spread of resistance

Question 27

How can E. coli be beneficial?

Answer 27

Forms part of the normal microbiota of the large bowel and possible protects against invasion from pathogenic species such as salmonella.

Question 28

What types of conditions can E. coli cause?

Answer 28

* intestinal infections (diarrhoea) * toxin-mediated disease * extra-intestinal infections: * Urinary tract * Intra-abdominal * Biliary tract * Bloodstream infection * Neonatal meningitis The propensity to cause disease is linked to the presence of virulence factors, frequently restricted to specific E coli strains

Question 29

Name the types E. coli that cause diarrhoea

Answer 29

``` Six pathotypes of diarrhoeagenic E coli • Enterotoxigenic E. coli(ETEC) • Enteropathogenic E. coli(EPEC) • Enteroaggregative E. coli(EAEC) • Enteroinvasive E. coli(EIEC) • Diffusely adherent E. coli(DAEC) • Shiga toxin-producing E. coli(STEC)—also called Verocytotoxin-producing E. coli(VTEC) or enterohaemorrhagic E. coli(EHEC). • EPEC and EIEC are most common among young children in the developing world. EAggEC are most common among immunocompromised persons ```

Question 30

What is Enterotoxigenic E coli (ETEC)?

Answer 30

* Important cause of bacterial diarrhoeal illness * Major cause of diarrhoea in lower-income countries, especially among children. * Leading cause of travellers' diarrhoea * Faeco-oral transmission * ETEC produces two toxins, a heat-stable toxin (ST) and a heat-labile toxin (LT). Although different strains of ETEC can secrete either one or both of these toxins, the illness caused by each toxin is similar * Toxins stimulate the lining of the intestines causing them to secrete excessive fluid producing profuse watery diarrhoea and abdominal cramping * Less common -Fever, nausea with or without vomiting, chills, loss of appetite, headache, muscle aches and bloating * Onset 1-3 days after exposure and usually lasts 3-4 days

Question 31

What is enteropathogenic E coli (EPEC)?

Answer 31

• EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (AE) lesions. EPEC has Type 3 secretion machinery which forms translocation tube across membrane of enterocyte. secretes a protein into enterocyte which is an intimin receptor which is produced by the EPEC which can bind to the TIR which has become localised to the cell membrane and anchors the bacteria to the cell membrane allowing the bacteria to start getting nutrients and cause damage to the cell. It subverts the cells own systems to cause disease. • The translocated EPEC proteins also activate signalling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria.

Question 32

What is Shiga toxin-producing E coli (STEC)?

Answer 32

* Also known verocytotoxic E. coli(VTEC) or enterohemorrhagic E. coli (EHEC) * Causes haemorrhagic colitis (bloody diarrhoea) and haemolytic uraemic syndrome (HUS -triad of acute renal failure, haemolytic anaemia, and thrombocytopenia) Produces bloody diarrhoea Toxin works by binding to intestinal cells and subsequently causing cell death and thereby damage to the lining of the intestine with bleeding.

Question 33

What is the molecular action of Shiga toxin?

Answer 33

* B sub-units of Shiga toxins bind to globotriaosylceramide(Gb3) on host cell surface * After binding, the toxin is endocytosed and transported to the Golgi apparatus and the endoplasmic reticulum * During the intracellular transport, the A chain is cleaved into the small A2 fragment and the enzymatically active A1 fragment. * The A fragments are kept together by a disulfide bond until the toxin reaches the ER, where the A1 fragment is released and translocated into the cytosol * In the cytosol, the A1 part inactivates ribosomes thereby inhibiting protein synthesis, eventually resulting in cell death.

Question 34

What are Extra-intestinal pathogenic E coli (ExPEC)?

Answer 34

* Strains of E coli capable of causing disease outside the intestinal tract * Wide range of virulence factors: * Adhesins - Help attach to the surface of cells * Iron acquisition systems - scavenge iron * Protectins and invasins - protect themselves from immune response. * Toxins * Others

Question 35

How does E.coli cause urinary tract infections?

Answer 35

Urinary tract infections are very common in women but unusual in men This difference is explained by anatomical differences between women and men Uropathogenic E coli transfer from the rectum to the urethra and then migrate to the bladder, causing cystitis (bladder inflammation). This journey is far easier in women than men due to shorter distance.

Question 36

What type of E.coli can cause UTIs? Explain how some of their virulence factors work

Answer 36

UropathogenicE coli (UPEC) Adhesins: • Type 1 fimbriae have adhesive tips that bind to α-D-mannosylated proteins on uroepithelium, mediating adhesion, invasion of uroepithelium and the formation of intracellular bacterial communities (IBCs) Toxins • Lipopolysaccharide (LPS) • α-Haemolysin(HlyA) is a secreted, pore-forming toxin, cytotoxic towards epithelial cells in the urinary tract Iron acquisition • The availability of iron is extremely restricted in the urinary tract • Bacteria produce their own iron-complexing proteins (siderophores) to acquire iron.

Question 37

Can E.coli cause bloodstream infections?

Answer 37

E coli bacteria are the commonest cause of bacterial bloodstream infection in England and the number of infections is rising. • Around half of E coli bloodstream infections occur in patients older than 75 years • Causal factors: • 50% recently had UTIs -possibly linked to ineffective antibiotic treatment??? • 21% of patients have urinary catheters • 16% hepatobiliary infections • 7% gastrointestinal infections The incidence of E coli bloodstream infection is around 50% higher in the north of England than the south of the country Incidence increasing in England with correlation with rising environmental relationship, also seasonal with more incidence in the summer?

Question 38

How do we manage E.coli infections that cause diarrhoea?

Answer 38

* PREVENTION * Avoid foods and drink that could be contaminated with bacteria: * Raw fruits and vegetables (e.g., salads), raw seafood or undercooked meat or poultry, unpasteurized dairy products, food from street vendors, and untreated water (including ice) in areas lacking adequate chlorination. * TREATMENT * Most infected persons will recover within a few days, without requiring any specific treatment. * Clear liquids are recommended for persons with diarrhoea to prevent dehydration and loss of electrolytes. * Oral rehydration solutions * Avoid antibiotics –may make illness worse

Question 39

How do we manage E.coli UTIs?

Answer 39

• In UK, antibiotic treatment of UTIs is largely empirical • Commonly used antibiotics are trimethoprim and nitrofurantoin • Around 60% of E coli urine isolates tested in laboratories are trimethoprim resistant

Question 40

How do we manage E.coli bloodstream infections?

Answer 40

* Increasing resistance to a wide range of antibiotic classes * Resistance genes frequently on plasmids, therefore horizontal gene transfer common * Around 40-50% of isolates resistant to co-amoxiclav * Variable but increasing prevalence of carbapenemase genes. * Resistance linked to sequence type