Dr. Fane
 Office: 102A VetSci / Micro Building
 Phone: 626-6634
 Email: bfane@u.arizona.edu

 
Objectives

The objective of this course is to introduce students to virology research and skills used for the study of viruses. The experiments in the course have not been conducted before and represent novel lines of investigation. Although preliminary hypotheses will be discussed, unlike most undergraduate laboratory exercises, experimental results cannot be predicted. They will either prove or disprove a hypothesis. Moreover, since many experiments are being conducted for the first time, problems may arise. Thus, the syllabus should be regarded as a tentative schedule.

Experiments and hypotheses to be tested


The evolution of viral resistance: Genetically modified external scaffolding genes have already been cloned. The external scaffolding protein, along with the internal scaffolding protein, mediate the assembly of the virus. The expression of these mutant external scaffolding proteins prevents wild-type virus propagation in infected cells. Viruses resistant to the expression of these inhibitory proteins have been isolated via direct genetic selection and have also been evolved in a laboratory setting by continual passage. Hypotheses to be tested: 1) Strains evolved to be resistant to the anti-viral proteins will in fact become dependent on them, exhibiting optimal growth only in the presence of the antiviral protein. 2) The mechanism of resistance involves altering the interactions between the viral coat protein and the internal scaffolding protein; in other words the participation of the internal scaffolding protein becomes more important. Several lectures will be dedicated to a more through explanation of these phenomena.


The Microviridae, a family divided: There appears to be a wide evolutionary chasm separating the two sub major subfamilies of the Microviridae. Members of the Microvirus subfamily (Micro being Greek for small) have been isolated from free living bacteria. The Gokushoviruses (Gokusho being Japanese for very small) have been isolated from obligate intracellular parasitic bacteria, such as Chlamydia and Bdellovibrio. Although Gokushoviruses have been isolated from widely different hosts, Chlamydia, Bdellovibrio, and Spiroplasma, Microviruses have only been isolated from closely related enteric bacteria. Hypothesis to be tested: Microviridae subfamily type is a function of the life-style of the bacterial hosts, free living v obligate intracellular.


Readings: Reading materials will be provided. Links to the reading material can be found in the syllabus.

Grading: There will be two one hour exams and a lab report, each worth 100 points. An additional 30 points will be assigned from laboratory notebooks. Exam questions will primarily be short answer, short essay, and data analysis questions. If the average of any given exam is below 75%, that exam will be curved with the average being set at 75%.

Attendance: If a student has more than one unexcused absence from the course. S/he will be dropped from the course.

 Grade calculation: A student can receive a total of 315 points in the course. Grades, however, will be calculated by dividing the points obtained by 300. No exams are dropped.

 Grading: 90%-105% A; 80%-90% B; 70%-80% C; Below 70% E. Note that the D grade is not used in this course.

Office hours: by appointment. However, both the TA and the professor will be in the laboratory. Often there is "down" time during experiments, which provides a wonderful time to ask questions.

Policy on missed exams and labs: Students representing the University in an official capacity, as defined by the Student Handbook, must notify me before the exam to schedule an alternative date. Students, who miss an exam or lab due to illness or other emergency, must contact me the day of the exam to schedule a make-up exam. A doctor's note may be required.

Preparation for laboratory: Students are expected to come prepared for the lab. This includes preparing an outline of all protocols.  If a student is not prepared, the TA and/or the professor has the authority to require the student to do this preparation during the laboratory, before continuing with that day's assignment. If the student is unable to finish the laboratory within the specified time period, no make-up will be given. 

Safety procedures: A list of safety procedures and lab rules will be circulated. Students are to read this carefully and sign the bottom of the page, which attests to their understanding of the safety procedures. If a student does not wish to sign this agreement, s/he will have to drop the course. If a student fails to abide by the safety rules, the TA and/or the professor has the authority to ask him/her to leave for the period.

Lab notebooks: Students must keep a lab notebook. The notebook is to contain protocol descriptions, to be done before lab, a brief description of the theories behind the protocol and data, to be collected during lab. Data may include drawings, written description, tables, and figures. Protocols and theories must be in the student’s own words.  Copying text verbatim from the lab notebook is not acceptable.

Policy regarding missed lab periods and exams: Students representing the University in an official capacity, as defined by the Student Handbook, must notify their TA before the exam and/or lab period to schedule an alternative date. Students who miss an exam or lab period due to illness or other emergency, must contact the TA the day of the exam or lab period to schedule a make-up. A doctor's note will be required. No make-up labs will be given for unexcused absences. For each unexcused lab session, 5 points will be subtracted from the total points obtained by the end of the course. These policies are not designed to force students to be responsible.  That decision rests with the individual. These policies are designed to minimize the adverse effects an irresponsible individual has on others. The lab staff is extremely busy, concurrently operating laboratories for several courses. Their dedication and hard work is not to be taken lightly. A student who is consistently unprepared for lab has adverse effects on other students, most notably his/her lab partner. In addition, an unprepared student requires a disproportionate amount of time from the TA.  This hinders the TA from helping other students who have also paid tuition for this course. 

 

Syllabus:

Date	Lecture (50 minutes)	Lab
Day 1	Lecture 1: Viral life cycles.	Lab 1: Basic skills: plaque assays and serial dilutions.
	Lecture 2: The Microviridae.
	Lecture 3: Theory and practice of laboratory-based virus propagation, serial dilutions, plaque assays, moi, burst.
Day 2	Lecture 4: Principles of experimental evolution with viruses and fitness.	Lab 2: Skills for evolutionary passage experiment. Preparation of cell cultures for experimental evolution experiments.
	Lecture 5: Antivirals and viral resistance.
	Lecture 6: Hypotheses to be tested in the evolution experiments.
Day 3		Lab 3: Serial passages and selection of viral resistance mutants.
Day 4		Lab 3 continued.
Day 5		Lab 3 continued.
Day 6		Lab 3 continued.
Day 7	Lecture 7: DNA biochemistry I: PCR, DNA purification, cloning, sequencing, and agarose electrophoresis.	Lab 4: DNA biochemistry: PCR reactions for sequencing evolved strains and cloning.
	Lecture 8:  Complementation and recombination in viral systems.
	Lecture 9: Recombination rescue and the hypotheses to be tested in the rescue experiments.
Day 8		Lab 4 continued: Purification and restriction enzyme digestion of PCR products and vectors. Purification of DNA for sequencing.
Day 9		Lab 4 continued: Purification of DNA for cloning,  ligation  reactions. Preparation of competent cells.
Day 10	Lecture 10: Protocols for recombination rescue experiments.	Lab 4 continued: Transformations.
Day 11	Lecture 11: Exam I	Lab 4 Propagation of clones. Preparation of competent cells.
	Lecture 12: Scientific writing.
Day 12	Lecture 12: The theory and practice of biophysical separations and purifications,  large particles.	Lab 4:  Plasmid isolation and transformations.
Day 13	Lecture 13: The theory and practice of biophysical separations and purifications,  proteins	Lab 5: Recombination rescue experiments. Propagation of hosts for the recombination rescue experiments. Lab 6: Isolation of novel Microviridae by rate zonal and equilibrium sedimentation: rate zonal purification.
Day 14		Lab 5 continued: Recombination rescue experiments. Generating recombinants.
		Lab 6 continued: Equilibrium sedimentation.
Day 15		Lab 5 continued: scoring recombinants. Lab 6 continued. Virus isolation
Day 16	Lecture 14: Exam II	Lab notebooks due, lab reports due. Finish experiments.