Monday, 28 January 2008

MMIC PBL - part 2 (avian flu)

Avian Influenza (Bird Flu):


Influenza viruses are segmented, negative-sense, single-stranded RNA viruses of the family Orthomyxoviridae and are divided into A, B and C.

Avian Influenza or bird flu refers to the different strains of influenza virus adapted to birds, a specific host. This is known as Influenza A virus, whereby all subtypes are adapted to birds. Generally, most avian influenza viruses are noninfectious for most species of birds, and are usually asymptomatic (no symptoms) if infectious. Hence, they do not pose much of a threat despite being highly transmissible.

However, domestication of birds/poultry has produced subtypes of avian species that are vulnerable to the viruses that rapidly mutate, causing many bird-related deaths. These become dangerous when the virus mutates and is transmissible to humans, such as the H5N1 virus. Other such viruses includes the H1N1 (Spanish flu) and the H9N2 (Hong Kong flu). Alternatively, viruses may mutate and infect an intermediate host such as the pig/swine, which support reassortment of genes that create new subtypes, before going on to infect humans.

As of 2004, Indonesia has had outbreaks of the H5N1 virus – avian flu. This strain is deadly to humans and usually spread through contact with poultry and domesticated birds like the fighting cock in Thailand. Indonesia has had outbreaks of H5N1 viruses, mainly from the poultry breeding farms. Typically, H5N1 transmission is restricted to from bird to humans. However in 2006, there was a case of human to human transmission of H5N1 versus between a group of small families, but the spread appears to have died off. H5N1 can last indefinitely at a temperature dozens of degrees below freezing.

Hence, this blog will concentrate on H5N1 virus as it is the most relevant in indonesian context as well as given time, and threatening to human life.

Strains of Influenza A:
Subtypes of influenza A as previously mentioned are based on antigenic relationships of the hemagglutinin (H) and neuraminidase (N) surface glycoproteins, two proteins on the surface of the virus that allow it to enter and exit host cells.
o Each virus has one HA and one NA protein, potentially in any combination
o Sixteen different hemagglutinins (H1-H16) and nine different neuraminidases (N1-9) have been identified to date.
o Typically, most avian flu is restricted to bird to bird transmission. The most deadly being H5N1 strain as it can spread from bird to human. > wiki > Flu_und_legende_color_c.jpg


Virulence is determined based on antigenic properties of the strain.
Highly pathogenic avian influenza (HPAI) viruses causes systemic disease with rapid death rate, mostly of the H5 or H7 subtypes
Low pathogenic avian influenza (LPAI) viruses cause a localized infection with little or no disease.
Virulence factor is also correlated with the hemagglutinin cleavage site – all HPAI have multiple basic amino acids (arginine and lysine) at the HA0 cleavage site, while LPAI have 1 or 2 amino acids. Multiple amino acid sites allow for a wider range of protease cleavage, resulting in higher amounts of reassortment, increasing virulence and susceptibility in other animals. This also allows for host protease to act on HPAI, causing it to replicate systemically, damaging vital organs and tissues, which results in severe disease and death as seen in H5N1.

Spread to humans via contact, potentially airborne (incubation period is longer, less adapted to droplet transmission):
- Directly from birds or from avian virus-contaminated environments to people (consumption of poultry products, direct contact with live poultry – bodily fluids such as blood, salvia etc and contaminated food sources).
- Through an intermediate host, such as a pig.
Typically develops 1 to 5 days following exposure.

- H5N1 virus particles enter blood stream through contact with chickens/ poultry. Virus attaches to cell surface sialic acid via receptor site on the haemagglutin (HA) site. Adhere to endothelium in respiratory tract.
- Internalised by receptor mediated endocytosis within cellular endosomes
- Viral envelope and cell membrane fuses, releases viral particles into cytosol.
- Cytosol acidic pH causes conformational changes in HA structure to form HA2, that binds to membrane to open M2 ion channel.
- Ions from endosome enters virus particle, triggering another set of conformational changes to HA, releasing viral nucleocapsids into the cell cytoplasm.
- Viral transcription occurs in the nucleus by viral encoded polymerase, consisting of a complex of 3 P proteins. This is activated by RNA polymerase II that caps and methylates the 5’ terminal.
- Viral proteins such as NS and NP are synthesized
- NS proteins interact with M proteins for nuclear export of viral RNPs.
- Templates for viral synthesis remain coated in nucleocapsid which are neither trunucated or methylated
- Viral mRNA are synthesized by viral encoded polymerase using positive template strand.
- Individual viral components arrive at budding sites by different routes
- Nucleocapsid is assembled in the nucleus and moves out to cell surface
- Glycoproteins HA and Na are synthesized in the endoplasmic reticulum, modified and inserted into the plasma membrane
- M protein serves as a bridge, linking nucleocapsid to sytoplasmic ends of glycoproteins
- Progeny virons bud off from cell when HA is cleaved to HA1 and HA2 by proteolytic enzyme from host. NA removes terminal sialic acid from cellular and viral surface viral proteins, facilitating release of viral particles from cell.

Signs and symptoms:
- Cough
- High fever (typically > 38°C)
- Headaches
- myalgia (muscle ache/pain)
- malaise (general discomfort)
- Sore throat
- Shortness of breath
- Diarrhoea
- A relatively mild eye infection (conjunctivitis), sometimes the only indication of the disease.

Severe signs and symptoms:
- Viral pneumonia
- Acute respiratory distress (the most common cause of bird flu-related deaths)
- Seizures

Laboratory Diagnosis:

Samples to be taken: Nasal washings, gargles, throat swabs. Within 3 days of onset of symptoms.
Sample conditions: Held at 4oC until inoculation into cell culture (freezing/thawing reduces viral recovery).

Isolation methods: embryonated eggs, monkey kidney cells.

1) Viral isolation
Viral isolates can be identified by hemagglutination inhibition, that rapidly determines influenza type and subtype. Test serum/culture fluid with hemagglutination inhibitor to check for presence. If results are negative, make a passage into fresh culture as influenza is typically fastidious and grows slowly.

2) Fluorescent antibodies
For rapid diagnosis, cell cultures on coverslips may be inoculated and stained 1 or 2 days with monoclonal antibodies to respiratory agents. Positive confirmed with use of single fluorescent antibody.
Alternatively, a more rapid diagnosis with less sensitivity is directly using fluorescent antibody on nasal aspirate.

Sample: Blood, serum.

3) Serology
Normal individuals will produce influenza antibodies during infection, such as antibodies to hemagglutinin, neuraminidase, nucleoprotein and matrix. These can be deteced by ELISA or hemagglutination inhibition (HI). These are performed using the patient’s serum extracted from blood stream. ELISA is more sensitive than other assays, while HI enables pin pointing of influenza strain. However, this method is dependant on patient’s antibody response system.

Necessary precautions:
1) Avoid contact with live birds, and all forms of poultry:
- Chickens, ducks, turkeys and geese and their feces, feathers and pens if at all possible.
2) Avoid poultry products in Indonesia, as cases of H5N1 have been reported there.
3) All foods from poultry, including eggs should be cooked thoroughly. Egg yolks should not be runny or liquid. Influenza viruses are destroyed by heat, hence cooking temperature for poultry meat should be 74oC (165 F).
4) Avoid cross contamination of other foods by use of separate kitchen utensils and surfaces exposed to raw poultry.
5) Wash hands with soap and water after any poultry contact.
6) Avoid live food markets.

Suppress virus, keep it from replicating and infecting within the host. Must be taken soon (often within 48 hours following infection).
Neuraminidase inhibitor:
Mode of action consists of blocking the function of the viral neuraminidase protein, preventing the virus from reproducing.
1. Relenza
2. Tamiflu

Vaccines expose an individual to a weakened/dead virus to stimulate antibody production against it, so that immune system can fight off infections should it arise.
There are at least 15 different strains of avian flu and they are constantly mutating, hence vaccination may not prove to be effective for long.
Live vaccines (attenuated, weakened):
- Requires less antigen (active ingredient) than killed vaccine.
Live vaccine may contain too few copies of the weakened virus to trigger an immediate immune response.
However, once inside the host, the virus can replicate to render it detectable by the immune system and trigger an immune response.
Does not require injection – oral consumption will do.
Killed virus (inactivated):
Must be injected – only route to administer them that will bring them into contact with the immune system.
Requires larger dosage than live vaccines due to its lack of ability to multiply within host.
3. Recombinant vaccine:
Genetically engineered vector (usually a low virulence virus) to express H5N1 protein antigen on surface, to stimulate immune response (production of antibodies).

References: > releases > 2007 > 08/070828154944.htm > cidrap > content > influenza > avianflu > news > dec3005halvorson.html
evolution > > evolibrary > news > 51115_birdflu> news > background > avianflu > protection.html > ncidod > EID > vol10no4/03-0396.htm
Debra, TG02

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