Background: Influenza virus causes respiratory infections (“the flu”) that can be quite severe. About 36,000 people die from flu complications every year in the U.S. The symptoms of the flu include fever, headache, sore throat, cough, and muscle aches. These symptoms are difficult to distinguish from other respiratory infections. A more definitive diagnosis can be made by testing respiratory fluids, often obtained via a throat or nasopharyngeal swab, for the presence of influenza proteins (using ELISA or similar techniques) or nucleic acid (using RT-PCR) (Since the virus genetic material is RNA, reverse transcription (RT) is used to copy the RNA into cDNA for the PCR test). Since it takes a few weeks for antibodies to influenza to be detected in a patient’s blood, testing the patient’s blood for influenza antibodies is usually used to confirm an infection after the illness has subsided, e.g. to monitor the extent of an epidemic. Influenza virus is highly contagious and is easily spread via respiratory droplets.
There are drugs available to treat influenza virus infections, but they are generally only used when an infected person is at risk for serious complications, or to control an epidemic. The influenza vaccine (“flu shot”) can protect individuals from getting infected, and it is highly recommended for individuals most at risk for the serious complications from flu, such as people older than 65, children under 2 years old, and anyone with chronic heart or lung conditions. The vaccine usually contains three different strains of influenza, and the antibodies generated to the vaccine can protect against these strains and related strains. Influenza virus has a high mutation rate and there are new strains of the virus appearing every year. There are two main strains of influenza, A and B, that cause the annual outbreaks of flu. Strain A viruses are further distinguished based on their surface proteins, abbreviated H and N. The strain names also include the location and year where they were isolate. For example, strain A/Fujian/02 (H3N2) was isolated in the Fujian province in China in 2002 and its surface proteins are designated H3N2.
Note: An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control value (the ELISA tutorial provides details of the procedure). PCR results are recorded as viral load values, i.e. how many copies of the virus were detected. Use the 96-well PCR feature to analyze these cases, including the determination of viral load.
Case A. This fall, for the first time in several years, Sheila did not get a flu shot. She has been very busy, especially since she started babysitting her grandchildren (ages 1 and 3) on weekdays. She also does not like needles and shots, so it was easy for her to come up with excuses not to go get the shot. Sheila is 67 years old, but she has been in good health and does not have any chronic health conditions. Two days ago, she came down with a fever (102 degrees F), sore throat, and a bad cough. She has been taking ibuprofen, but it does not seem to be helping. Sheila feels just awful, but she drags herself to the clinic. The physician is concerned that the fever has not subsided, and because Sheila’s age places her at some risk for serious complications from influenza, she decides to test Sheila for influenza and takes a throat swab sample. Although Sheila’s grandchildren have not been with her the past two days, they did stay at her house the day before she got sick. The physician suggests that both children be tested, even though they have not yet shown any symptoms.
Procedure: To analyze this case, first run an ELISA on the throat swab samples from the following sources to test for the presence of influenza virus proteins, using antibodies specific for influenza A and B viruses. Optionally, you can perform a Western blot on each sample to confirm the ELISA results. Then perform a 96-well PCR test on the cDNA isolated from the swabs, using primers specific for influenza A and B, to see if influenza virus genetic material can be detected. Use the 96-well PCR format for this test.
Samples for ELISA
- Protein: Negative control (no influenza virus)
- Protein: Positive control for influenza A
- Protein: Positive control for influenza B
- Protein: Sheila
- Protein: 1-year-old
- Protein: 3-year-old
- Antibody: Positive for influenza A
- Antibody: Positive for influenza B
Samples for 96-well PCR
- DNA: Negative control (no influenza virus)
- DNA: Positive control for influenza A
- DNA: Positive control for influenza B
- DNA: Sheila
- DNA: 1-year-old
- DNA: 3-year-old
- Primers for influenza A
- Primers for influenza B
- Do Sheila’s symptoms appear to be caused by infection with influenza virus?
- Are either of the grandchildren infected?
- What should the physician recommend for treatment for these patients?
- What should Sheila do to avoid spreading her illness to others?
Bioinformatics: Influenza B viruses generally respond well to the antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relenza), which inhibit neuraminidase activity. However, influenza B viruses have been isolated this season that show some resistance to oseltamivir. Compare the neuraminidase sequence isolated from Sheila’s virus to these resistant virus isolates as well as to some drug-sensitive viruses. Does it look like oseltamivir will be an effective treatment for Sheila? What other options does she have? [Note: Use the sequences in the Bioinformatics folder, rather than the PCR folder, for this analysis.]
Case B. Shannon, a college junior, was really looking forward to playing in her first big basketball tournament. The whole team had flow to Hawaii for one week during the semester break to play against teams from all over the country. Unfortunately, Shannon’s team was not in top form. Three team members had to stay home due to illness. They had headaches, fever, and muscle aches that prevented them from getting out of bed, let alone play basketball. Four other teammates had similar symptoms the week before but had recovered enough to join the team on the trip, although they were somewhat out of practice. On the morning of their first game, Shannon woke up in the hotel room she was sharing with three other teammates feeling terrible. She was distressed to realize she was experiencing the same symptoms as the sick team members, and she did not want to tell her coach because she did not want to miss the game. But soon after getting up she knew she was too sick to play and reluctantly told her coach. The coach was anxious to find out what was sweeping through her team, so she brought Shannon to a clinic in Honolulu. The physician there took a throat swab and tested for influenza virus. She also recommended that the other team members rooming with Shannon be tested. In addition, she tested blood from the team members who had recovered from a similar illness, to see if they had antibodies against the same virus.
Procedure: To analyze this case, run an ELISA on the throat swab samples from the following sources to test for the presence of influenza virus proteins, using antibodies specific for influenza A and B viruses. Then perform a 96-well PCR test on the cDNA isolated from the swabs, using primers specific for influenza A and B, to see if influenza virus genetic material can be detected. Use the 96-well PCR format for this test.
Samples for ELISA
- Protein: Negative control (no influenza virus)
- Protein: Positive control for influenza A
- Protein: Positive control for influenza B
- Protein: Shannon
- Protein: Roommate 1
- Protein: Roommate 2
- Protein: Roommate 3
- Antibody: Positive for influenza A
- Antibody: Positive for influenza B
Samples for 96-well PCR
- DNA: Negative control (no influenza virus)
- DNA: Positive control for influenza A
- DNA: Positive control for influenza B
- DNA: Shannon
- DNA: Roommate 1
- DNA: Roommate 2
- DNA: Roommate 3
- Primers for influenza A
- Primers for influenza B
Procedure: Test the blood samples from the four players who recovered from the illness using an ELISA to see if they have antibodies to influenza A or B proteins. Note for this test, the wells are coated with proteins because the goal is to look for antibodies in the sample (the reverse of the previous procedure), so make sure to select that option when you use the autoload feature.
Samples for ELISA
- Protein: Positive control for influenza A
- Protein: Positive control for influenza B
- Antibody: Positive for influenza A
- Antibody: Positive for influenza B
- Antibody: Negative control
- Antibody: Recovered player 1
- Antibody: Recovered player 2
- Antibody: Recovered player 3
- Antibody: Recovered player 4
- Is Shannon infected with Influenza virus?
- What are Shannon’s prospects for playing in any of the games this week?
- What should the physician recommend for Shannon?
- Are any of her roommates infected?
- Does is appear that the recovered players had the same virus infection?
- What should be done to prevent the rest of the team from getting sick?
- Should 96-well PCR have been run for the recovered players, in addition to ELISA?
Bioinformatics: A novel variant of H1N1 influenza, with a possible origin in swine, has been reported in Mexico and part of the U.S. So far, it has not been detected in Hawaii. But since the antibody testing does not determine which type A influenza Shannon and her roommates have contracted, hemagglutinin sequences from their virus isolates are compared to the novel H1N1 virus, as well as to seasonal H1N1 viruses that have been circulating in Honolulu the last two years. Do Shannon and her roommate appear to be infected with seasonal flu or with the novel swine flu? Does it matter in terms of their treatment? [Note: Use the sequences in the Bioinformatics folder, rather than the PCR folder, for this analysis.]