Labeling techniques for immunofluorescence microscopy and flow cytometry Kuby, Kindt, Goldsby, Osborne Immunology Textbook
Flow cytometry -- F luorescence A ctivated C ell S orting (FACS) A two-color FACS analysis Kuby, Kindt, Goldsby, Osborne Immunology Textbook Single color FACS analysis (e.g., using anti-A antibody) -- note this is a log scale A- cells A+ cells
Stanford used flow cytometry to screen blood before HIV tests were available
Reduced ratio of CD4+ to CD8+ T cells in AIDS patients
July 1983 to June 1985, Stanford Blood Center used flow cytometry to test donated units for CD4:CD8 ratio
Did not transfuse blood from donors with CD4:CD8 ratio < 0.85
Most other blood banks did no screening
~10,000 cases of transfusion-transmitted AIDS in US before HIV test available in 1985
Galel et al., 1995, “Prevention of AIDS transmission through screening of the blood supply” Annu. Rev. Immunol. 201-227
Swine flu is a dangerous form of influenza. Which of the following is true:
Swine flu is highly contagious amongst pigs, but does not infect humans.
Swine flu is normally spread only amongst pigs, but can be caught by humans by eating infected pork products.
Swine flu can be caught by humans through contact with infected pigs and/or their environment. However, there are NO documented cases of human-to-human spread.
Swine flu can be caught by humans through contact with infected pigs and/or their environment. Many of the current swine flu cases resulted from human-to-human spread.
Clicker question Bird flu is a dangerous form of influenza with a high fatality rate. Which of the following is true: 1) Bird flu was created accidentally by making flu vaccines in hen eggs. 2) Bird flu is highly infectious in birds, but does not infect humans. 3) Bird flu can infect humans and birds. Humans are infected only through direct contact with infected birds (no human-to-human transmission). 4) Bird flu can infect humans and birds, but there are very few documented cases of human-to-human transmission. Only a few mutations could convert it so that it is easily transmitted between humans. 5) Bird flu can infect humans and birds. There are currently many documented cases of human-to-human transmission. A vaccine against bird flu is already available and is responsible for reducing the number of human-to-human transmissions of bird flu.
M1 Matrix protein Hemagglutinin Neuraminidase M2 ion channel Viral ribonucleoprotein Nucleoprotein Viral RNA 1. Virion enters cell by receptor-mediated endocytosis. Taken into endosomes. 2. Low pH allows fusion. M2 ion channel makes inside of virus more acidic. M1 dissociates and viral ribonucleoproteins enter nucleus. 3. Viral polymerases transcribe (3a) and replicate (3b) the RNA. 4. Newly-synthesized mRNAs go to cytoplasm, where they are translated. 5. Post-translational processing of viral proteins in Golgi (5b). Nucleoprotein goes to nucleus (5a). 6. Nucleoprotein binds new RNAs, go to region of cell surface with H, N and M2. 7. Budding. http://www.ncbi.nlm.nih.gov/genomes/FLU
Infection by influenza virus Host cell receptors (sialic acid-containing glycoproteins) Flu viruses get taken into acidic endosomes by receptor-mediated endocytosis. Low pH induces fusion of the flu virus membrane with the endosomal membrane, resulting in release of viral RNA into the cytoplasm. HIV fuses at the surface of the host cell -- doesn’t require low pH for fusion. pH 7.4 pH ≤6.0
Influenza virus hemagglutinin (H or HA) binds to sialic acids on host cell glycoproteins
Flu virus H is a trimer. Each subunit contains two chains: HA1 and HA2.
HA1 (gray) contains the sialic acid binding site. HA1 is the counterpart of HIV gp120 .
HA2 (colored) is responsible for fusion. HA2 is the counterpart of HIV gp41 .
Sialic acid is the host cell receptor ( counterpart of CD4 ).
Original H structure: Wilson et al., 1981, Nature 289: 366-373 Hemagglutinin trimer Sialic acid Sialic acids are components of complex carbohydrates on cell surface glycoproteins. Different hemagglutinins are specific for different isoforms of sialic acid, hence defining host range of flu viruses.
Nature Structural & Molecular Biology 16, 233 - 234 (2009) Antibodies bind around the receptor binding pocket, but can’t access it completely (Canyon Hypothesis).
Hemagglutinin (H) undergoes a large structural rearrangement when exposed to low pH H monomer at neutral pH: fusion inactive H monomer at acidic pH: fusion active Receptor binding site Fusion peptide Fusion peptide Viral membrane Viral membrane Acidic pH: fusion active Note that predicted position of viral membrane has changed. Neutral pH: fusion inactive Fusion peptide
Residues in the hemagglutinin and HIV fusion peptides are primarily
Carr and Kim, 1993, Cell 73: 823-832 A spring-loaded mechanism for the conformational change of influenza hemagglutinin HA1 HA1 fusion peptide fusion peptide Viral membrane HA2 HA2 Note that hemagglutinin, like gp120/gp41, is actually a trimer -- what is shown schematically is the structure of a monomer. Analogy to fusion by HIV: HA1 is the counterpart of gp120 . HA2 is the counterpart of gp41 . The trigger for hemagglutinin for changing to its fusion-active form is low pH. The trigger for HIV spike protein for changing to its fusion-active form is binding to CD4 and CCR5 . Conformational changes also alter position of viral membrane -- not shown here.
Clicker question Influenza is an enveloped virus that buds out from the host cell membrane, incorporating flu proteins, including hemagglutinin, into its envelope. Influenza hemagglutinin binds to sialic acid, which is a component of N-linked carbohydrates attached by host cell glycosylation machinery. How does flu virus manage to bud from a cell that includes lots of sialic acid-containing proteins at its cell surface? (Wouldn’t it get stuck?) 1) Flu virus encodes an enzyme that cleaves sialic acids from host and viral proteins. 2) Once inside an infected cell, flu virus shuts down host cell machinery for attaching sialic acids. 3) Newly-budded flu viruses remain attached to host cells via interactions between hemagglutinin and sialic acid-containing host cell proteins. 4) Fevers that accompany influenza infection promote release of newly-budded flu viruses from host cells.
Neuraminidase monomer with bound inhibitor Influenza neuraminidase is an enzyme that cleaves sialic acids so that budding influenza viruses can be released from host cells Original neuraminidase structure: Varghese et al., 1983, Nature 303: 35-40
Palese, 2004, Nature Medicine 10: S82-S87 Inhibition of influenza replication cycle by antivirals
Two classes of FDA-approved drugs available against influenza A:
M2 blockers (e.g., amantadine, rimantadine ) prevent influx of protons through M2 ion channel, preventing release of viral genes into cytoplasm.
Neuraminidase inhibitors (e.g., oseltamivir (Tamiflu), zanamivir ) prevent release of virus from infected cell.
Drug resistance has been reported for both antiviral classes.
Swine flu (H1N1) is resistant to amantadine and rimantadine (M2 blockers) Swine flu is sensitive to oseltamivir (Tamiflu) and zanamivir (Neuraminidase inhibitors)
Hybrid viruses produced in a secondary host -- result from reassortment of segmented RNA genomes
Antigenic shift is rare, but shifted strains cause the flu pandemics that occur every 10-50 years.
What is the “secondary host”?
Antigenic shift in Type A influenza Avian versions of influenza A cause digestive tract infection in birds. Pig can be host to both avian and human influenza A. Some hybrid viruses can infect humans. Reassortment of viral gene segments most likely in places where pigs, ducks, and humans live in close contact.
Sequences of 2009 H1N1 Flu http://www.ncbi.nlm.nih.gov/genomes/FLU Also includes links about flu virus biology Early reports said 2009 swine flu was mixture of human, bird and pig influenzas. Later reports said 2009 swine flu is a reassortment from two pig flu viruses (North American and Eurasian swine flu viruses). The N1 gene is from Eurasian swine flu -- never before seen in humans.
Most flu viruses infect birds. “Bird flu” refers to influenza in birds and in humans in cases in which avian viruses cross the species barrier to infect humans.
2003-2004: Outbreaks of H5N1 in poultry in 8 Asian countries and 100 cases of human infections with 50% fatalities.
Few (maybe none) cases involved bird-to-human transmission. A pandemic would require human-to-human transmission. A change of only two amino acids would allow H5 to efficiently recognize human cells.
H5N1 viruses are being tested for making a vaccine, but mass production of an H5N1 vaccine would require safety and efficacy trials that might take longer than viruses would spread in a pandemic.
Antiviral drugs oseltamivir (Tamiflu) and zanamivir, but not amantadine, are active against H5N1. Supply not adequate for a global pandemic. Antiviral resistance occurs in N1 viruses during Tamiflu treatment.
Response to bird flu threat in Asia Protect domestic ducks Burn chicken carcasses Vaccinate chickens from contact with wild ducks http://pr.caltech.edu/periodicals/CaltechNews/articles/v41/uneasypieces.html