1. Decoding the Genetic Code Influenza Virus

2. Influenza - Symptoms

3. Influenza – The spread 3 The virus spreads easily by coughing and sneezing Influenza virus particles dry out (half-life a few hours at room temperature). Cold and dry weather allows the virus to survive longer outside the body than in warm weather. Once infected it takes 1-3 days to get sick. In humans, the virus infects cells lining the respiratory tract.

4. Influenza - Types Influenza AInfluenza B Influenza C

5. ‘Getting the Flu’ 5 Just about everyone has been sick with ‘the Flu’ at some stage in their lives. So, why do people get sick again and some die when they ‘catch the Flu’ again? Shouldn’t they have immunity to the Flu virus?

6. Antigens identify Flu strains 6 There are two types of antigen = N and H. In different virus strains, the shapes of N and H are different. There are 9 known N and 16 known H types. Influenza viruses are named according to the antigens (proteins) sticking out of their virus coat. (H) (N)

7. The role of antigens 7 The H antigen is like a key that allows the virus to enter into cells with a matching lock. This allows the virus to replicate inside the cell. Bird Flu H allows the virus to infect bird intestinal cells. Human Flu H allows the virus to infect human lung cells. The N antigen is required to cut the virus away from the host cell so it can spread to infect more cells. The N shown above has its cutting site blocked by a drug designed stop the flu from spreading.

8. Reaction to antigens Immune response Body produces antibodies against specific antigen If the antigens changes slightly (<1% = genetic drift), it can still be recognised quite quickly and your immune system will fight fast. You may be sick for a few days. If the antigens changes radically (genetic shift = up to 50%), it is not recognised. it takes longer for your immune system to prepare for war. The virus takes hold and can make you very sick. Major changes to the shape of the virus ‘face’ can cause a Pandemic

9. Pandemics – The Spanish Flu 1918 The Spanish Flu pandemic killed more than 40 million people! The virus antigens were extremely different to those encountered previously. People carried no immunity to this virus strain so they were highly susceptible to illness and even death. It started in America and then spread around the world with soldiers going to war.

10. What causes a Pandemic? 10 Genetic Shift! Let’s use the H5N1 Bird flu as an example…. H5N1 bird flu occasionally infects humans but at this stage humans do not pass this infection on to other humans. The spread of infection in birds means more humans will come into contact with and be infected by H5N1 bird flu. The concern? Eventually a pig will be infected with a human flu and a bird flu at the same time. They will serve as a ‘mixing pot’ for the two flu types to swap genes. The Result? A new flu subtype can emerge which easily spreads from person to person. An influenza pandemic would then occur with severe symptoms, like the lethal leakage of fluid into the lungs caused by the 1918 Spanish flu. This process of repackaging of viral genes is called reassortment. It is illustrated in the next slides.

11. Reassortment 11 Influenza A infecting a human. Can spread from human to human due to H and N proteins on surface. Influenza A infecting a chicken. Can occasionally infect humans but cannot spread from human to human due to H and N proteins on surface. Pig can become infected easily with bird flu and/or human flu. Serves as a mixing pot!

12. Process of infection and reassortment Virus coats break down and RNA genes move to the cell nucleus to be copied and transcribed. Viral genes are copied and prepared for packaging into new virus particles. Repackaging of genes creates a virus that can now transfer from human to human! PANDEMIC?

13. Naming the Flu 13 Try some yourself: A/swine/Ehime/80(H1N1)A/Tokyo/67(H2N2) A/duck/Hainan/2004(H6N2)B/Nanchang/97 NB. Occasionally you will find more in the name. For our purposes today, ignore those letters &/or numbers Answer question 3 in your worksheet A/chicken/Korea/01(H9N2) Influenza A virus was isolated from a chicken in Korea in 2001. The antigen types were H9 and N2

14. The Influenza Genome 14 All of the genetic material found in the virus is known as its genome. The genome is divided into 8 ribonucleoprotein (RNP) segments. The genetic material is (-) sense RNA (this is complementary to mRNA) Source: http://www.omedon.co.uk/influenza/influenza/

15. Influenza has 8 gene segments Sequencing has revealed the genome for influenza along with the proteins it codes for. SegmentSize (nucleotides) PolypeptideFunction 12341PB2Subunit of polymerase: Host cap binding and endonuclease 22341PB1Catalytic subunit of polymerase 32233PASubunit of polymerase, active in vRNA synthesis 41778HAHaemagglutinin 51565NPNucleoprotein: Part of transcriptase complex 61413NANeuraminidase: release of virus 71027M1Matrix protein: Major component of virion M2Integral membrane protein: Ion channel 8890NS1Anti-interferon protein. Effects on cellular RNA transport NS2RNP nuclear export 15 Source: http://www.omedon.co.uk/influenza/influenza/

16. The importance of Flu Chasers…. 16 Scientists monitor the Flu viruses circulating in the population by looking at changes to the virus H and N antigens. To identify the strain of flu virus…… Send a sample off to the laboratory for Gene Sequencing. This is an accurate way to find out the sequence of nucleotides in the viral RNA. After sequencing the H and/or N genes they can compare them with the gene sequences from other strains of the virus. This lets them look for the mutations that can cause epidemics and pandemics.

17. Mutant gene = Mutant Protein? 17 But does a change or mutation in the gene sequence always mean there will be a change in the protein or antigen? To work this out we must determine the gene sequence and then the amino acid sequence for the protein.

18. What is Gene Sequencing? 18 Gene sequencing is identifying and determining the order of the base pairs in a segment of RNA or DNA You will learn more about how scientists do this is a few weeks

19. 19 Copyright ©2000 by the National Academy of Sciences Reid, Ann H. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 6785-6790 The 1918 Spanish Flu N Gene Sequenced and Translated The underlined sequence codes for the signal peptide. Boxed amino acids indicate potential glycosylation sites. Circled amino acids indicate the active site residues (3).

20. Gene Expression This is not how transcription happens in the influenza virus! The flu virus genome is RNA NOT DNA! But, it still makes mRNA and so we can use the sense DNA genetic code. 20

21. Transcription – copy DNA into RNA 21 Try to work out the following: 1. The second strand of DNA (complementary strand). Remember the base pairing rule in DNA, A pairs with T and G pairs with C. 2. The code in RNA after copying the 1 st DNA strand (the sense strand). Be careful. In RNA, T is replaced by U. An A in the DNA sense strand will see U added to the growing RNA strand. Write your answer down and then click to check your answer. DNA: AAT CTG GGG AAC TCG TTT CGC CCC CGA TTA GAC CCC TTG AGC AAA GCG GGG GCT UUA GAC CCC UUG AGC AAA GCG GGG GCU UUA GAC CCC UUG AGC AAA GCG GGG GCU mRNA:

22. 22 mRNA containing the genetic code copied from the (-) sense RNA virus genome moves into cytoplasm of the host cell. Ready for……. Translation

23. 23 Source: http://genetics.nbii.gov/Basic1.html 5’3’ TRANSLATION: 1.mRNA locks onto a ribosome. 2.The ribosome reads the mRNA message 3 bases at a time = CODON 3.Transfer RNA (tRNA) molecules carry amino acids. Each tRNA has an anti- codon that will only base pair with the correct codon on mRNA. 4.Base pairing occurs between mRNA and tRNA and the new amino acid is added to a growing chain. Anti-codon = 3 bases in tRNA Amino Acid Codon = 3 bases in mRNA

24. 24 Source: http://psyche.uthct.edu/shaun/SBlack/geneticd.html Back to page 28 A series of three nucleotides coding for an amino acid in DNA is a triplet Nucleotides in the genetic code correspond to sense strand DNA or mRNA.

25. The Genetic Code 25 Since RNA is constructed from four types of nucleotides, there are 64 possible codons (4x4x4). Three of these codons specify the termination of the polypeptide chain = STOP codons. That leaves 61 codons to specify only 20 different amino acids. Most amino acids have more than one codon. Exceptions to this rule are the START transcription amino acid Methionine (Met) and the amino acid Tryptophan (Trp) The genetic code is said to be degenerate. NB/ The sequences coding for the protein N in this activity are shown as sense DNA rather than mRNA.