Course Portfolio: M435 Tissue Culture and Virology Lab

Joyce Patrick

Introduction: Student Demographics: Class size – 24 students, 67% male, 33% female Class Rank- Junior or Senior Course placement within curriculum: Elective for students enrolled for either a B.A. or B.S. in Biology, Microbiology or Biotechnology M435 is a laboratory class for Junior and Senior Biology, Microbiology or Biotechnology Majors. It is like many other science labs; students have a lab manual with pre-designed laboratory experiments. There are worksheets with each lab experiment; questions on the worksheet involve reporting results and interpreting results to assess student understanding of each experiment. Homework assignments cover broader topics from the class and offer additional opportunities for students to practice applying concepts from the lab. For the last several weeks of class, the students have to use what they have learned throughout the semester to identify an unknown sample and then write a lab report of their findings. However, there is very little practice throughout the semester of the style of writing required for the final lab report. The syllabus states that the learning objectives for the course are to “introduce students to basic concepts and experimental techniques used in a virology laboratory”. And that at the end of the course students should “understand the theory and have practical experience to successfully work in a virology research laboratory” (Appendix Item F). The grading scale in the class is divided between the worksheets (~33%), homeworks (~27%), Exams (~27%), and lab performance and participation (~6%) and the final lab report (~6%). However, 30% of the lab performace grade (1.8% of overall grade) comes from performance on the writing exercises leading up to the final lab report (see below). Objectives: Based on my evaluation of examples of student lab reports from previous incarnations of this class and others like it, it is obvious that students struggle with writing lab reports.

Common errors can be broken down into a few key themes: 1) students include too much or too little detail about experiments or present the wrong information. 2) Ideas are not organized into a cohesive “story” about a line of scientific inquiry. 3) Reports are often too narrative, explaining a series of events, rather than focusing on the purpose of the line of experimentation. Most of the problems result from a mistaken identity in the audience of the papers. I suspect that most students write their reports with the “Instructors as evaluators” in mind as an audience, when they should actually be writing their

reports for a knowledgeable peer as the primary audience. The objectives then, of this intervention were to aid students in a) identifying the correct audience for their papers, and b) tailoring the content of their papers to match the defined audience.

Defining audience has been reported as a hurdle in student writing assignments in the sciences. Karen Curto and Trudy Bayer reported that student writers had difficulty identifying audience and tailoring information for a defined audience. In that study, they had students work on an oral presentation on the same topic as the written assignment. They found that presenting to an audience made the audience’s reactions more apparent to the speaker/writer, and that after presenting material to a live audience, students were better able to write for the same audience (Curto and Bayer 2005). Due to the organization of the Tissue Culture and Virology class, presentations were not possible, however, a series of exercises and instructor feedback opportunities were created to help students properly identify the audience for their written reports.

Design: At the beginning of the unknown identification module, students completed a survey in class asking them about their writing ability and experiences (Appendix Item A). They received a model article and an accompanying writing sample worksheet to help them outline a section of their results (Appendix Items B and C). Midway through the module, student turned in a draft of the materials and methods section and results section for one experiment. The drafts were evaluated to see if students were able to accurately assess their ability and to see if any of the previously identified errors are present (Appendix Item D). Students receive comments and instructor feedback on their drafts, as well as a second questionnaire which was handed in with the final report (Appendix Item E). Assessment: Student experience and confidence. Two surveys were given to students during the writing process, one prior to any writing exercises, and one filled out and turned in with their final lab report. In order to assess how much experience students had at writing lab reports, the first question on the pre-writing survey (Writing Survey #1 – Appendix Item A) was “How many times have you written a paper based on your own data?” Of 22 respondents, over half of the students had written more than 20 lab reports in their college career (Table 1). Table 1: Student responses to the prompt “How many times have you written a paper based on your own data? (i.e. a lab report)”. Twenty-two students completed the survey. # of Lab Reports

1-5

5-10

10-20

20+

Unspecified/unanswered

% students responding

9%

4.5%

4.5%

64%

18%

This was reflected in the answers to the second portion of the survey, which asked students to rate their abilities at various aspects of writing, on a 5-step scale, from 1 (Very confident/good) to 5 (No confidence/poor). ~70-90% of the

class rated their abilities as either a 1 or 2 for both content and style of writing (Figures 1 and 2; White bars).

Figure 1: Student Responses on a 1-5 rating scale of perceived ability or confidence. Students were asked to rate their ability at organizing the content of their reports, selecting the correct details and types of information and content to include, and organizing data into meaningful and useful tables and figures. Scale was from 1 to 5, with 1 being “Very Confident/Good” and 5 being “No Confidence/Poor”. White bars represent responses prior to completing any writing sample worksheets or drafts (N=22). Black bars represent responses after completing the final draft (N=17). I also wanted to gauge student confidence in writing abilities, as well as lead students toward being metacognitive of their abilities. Part 2 of the survey was designed to answer this question. Students were most confident about their abilities to include the correct level of detail and information in their reports (Figure 1, Selecting Content, white bars). However, more students expressed a lack of confidence in this area than any other area. In addition, students were very confident in their ability to write in scientific style, and but almost a third of the students claimed some skepticism in their ability to correctly use scientific terminology (Figure 2, white bars). Finally, selecting appropriate content was one of the most problematic areas in the first draft of the “Materials and Methods” section (Figure 4).

Figure 2: Student Responses on a 1-5 rating scale of perceived ability or confidence. Students were asked to rate their ability at writing purposeful and coherent sentences and paragraphs, correctly using scientific terminology, and selecting, understanding and citing references. Scale was from 1 to 5, with 1 being “Very Confident/Good” and 5 being “No

Confidence/Poor”. White bars represent responses prior to completing any writing samplworksheets or drafts (N=22). Black bars represent responses after completing the final draft(N=17).

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utlining with a model.O In order to get students to think about organizing and

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tudent metacognition of ability.

selecting the content for their lab reports, an annotated model scientific article was posted on the class website (oncourse). The article served as professionaexample of scientific writing, with important components of the article highlighted.For example, the parts of a proper results paragraph, such as subsection headings, context, hypothesis, and experiment and results sentences wereclearly labeled and marked (Appendix Item B). Accompanying the article wahandout, and in order to increase incentive to complete the handout and make a genuine effort, 10 points of the lab performance grade were linked to the handout. Students were asked to first read the article, and then answer a of questions about their own experiment. The answers to the questions could easily be converted into an outline for a paragraph in a lab report. For example, the first question was: “What aspect of viral biotested or what are you trying to learn about the virus? Formulate a one sentence goal or hypothesis that explains what is being tested.” Some students were able to formulate very strong sentences, such as, “In order to characterize an unknown virus, a suitable host cell type must be selected based on its permissivity (sic) to the virus and its CPE upon viral infection.” While otstruggled, writing result-based sentences such as, “The virus is unstable at lopH and under lipid disintegration.” Most students answered the question in the format “This experiment aims to…” or “We did experiment x to…” They were thetypes of sentences students had written all semester on the worksheets in the labmanual. While these latter formats answered the prompt, they needed some adjustment to work well in the final drafts. S In the next step of writing, the students had to

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write a draft of a portion of the lab report. They were to write the Materials and Methods paragraph and the Results section with accompanying figure for a single experiment they had performed. First, student responses on the pre-writing survey were compared with their performance on the written draft. Mstudents were able to accurately estimate their ability to organize information; however it is unclear if the format of the experiments and reports made organization implicit, and students may have performed differently on a lstructured assignment (Figure 3). Most noticeably, students overestimated thability to select the appropriate level of detail to include in their reports. This is consistent with previous observations that students fail to correctly identify the proper audience, and thus describe some aspects of the experiment in great detail, while leaving others completely unexplained (personal observations; Cuand Bayer, 2005; Stockton 1994). Finally, about half of students were able to correctly assess their ability to generate figures from their data, while a few grossly underestimated their abilities. Almost no students had difficulty with

writing style, use of terminology, or selecting references, and were able to identify this strength readily.

Figure 3. Student opinion of writing ability compared to instructor evaluation. Students were asked to rate their ability at organizing the content of their reports, selecting the correct details and types of information and content to include, and organizing data into meaningful and useful tables and figures. Responses were compared to instructorassessment of performance on a written draft and categorized as either student accurately estimating ability, overestimating ability ounderestimati

r ng ability.

Common errors in drafts. The students wrote a first draft of the “Materials and Methods” and “Results” sections for a single experiment that had been performed. Using an assessment rubric, the drafts were rated as “Good”, “Average” or “Needs Work” in the areas of writing style, content, and figure design (Appendix Item D). Areas needing the most improvement were identified based on the ratings of the drafts in the above criteria. For the “Materials and Methods” section, most students needed to work on improving the organization of information under appropriate subsection headings, as well as the including all the necessary steps in an experiment. It is interesting to note that this area (selecting detail) was judged by the students as one of the areas of most confidence and least confidence (Figure 2). For the “Results” section, most students also needed to improve their use of subsection headings, brevity in word choice, and explaining the rationale for their experiment. Finally, students were able to design figures that accurately represented their data, but failed to include accurate labels, legends and descriptions (Figure 4).

Figure 4. Student performance on first draft. Drafts of “Materials and Methods” and “Results” sections, including a data figure for a single experiment were graded using a rubric (Appendix Item D). Students were rated as “Good” (white bars); “Average” (grey bars); and “Needs Work” (black bars). Improvement of the final drafts. After receiving feedback on the drafts, students were given the option of turning in a second draft of the same section, or drafts of other sections for more comments. These sections were not graded with the rubric, however, in-text comments were provided. Fifty percent of the class asked for additional comments on other sections or second drafts. Several students met individually with an instructor for one-on-one feedback and work on the drafts. Final drafts were turned in at the end of the semester and were graded using the same rubric as the draft. Scoring between the draft and the final were compared for each student, and improvement was noted as either no change, one step, or two steps. Moving up one step means a score change from “Needs Work” to “Average” or from “Average” to “Good”, and moving up two steps indicates a score change from “Needs Work” to “Good” on each parameter scored (Figure 5). In the “Materials and Methods” section, students showed the most improvement on organizing topics into subsections, and providing enough detail to make the experiment replicable by a knowledgeable peer. In the “Results” sections, students were able to improve brevity in writing, while simultaneously improving statements of experimental rationale and stating important controls and variables. Finally, figures were better labeled and described in the final reports than in the drafts.

Figure 5. Student Improvement on Final Lab Reports by Parameter Scored. Final lab reports were scored using the same rubric as the drafts. Each students performance on each parameter was counted as no change (White Bars), one step improvement from “Needs Work” to “Average” or from “Average” to “Good” (Grey Bars), or two step change from “Needs Work” to “Good” (Black Bars). Results are the comparison of the draft section to overall performance on the entire final report. Changes in student writing. As the final question of the post-writing survey, students were asked what changed most significantly about their writing between the first and final drafts. Out of 17 students completing the final survey, 5 mentioned making their final draft more concise, 8 mentioned organization, 9 altered the level of detail, 7 improved their writing style, and 4 improved the figures and tables presented. Analysis and Reflection:

I was surprised by the number of lab reports that students had written in the past. However, one student commented that the format we requested was not one she had written before. It would be interesting to use the pre-writing surveys for a number of years and look for a correlation between how many lab reports a student has written and writing ability. In the future, I would change several aspects of the Writing Sample worksheet. I would make it more explicit that it is an outlining exercise. Some students were answering as they would on a worksheet or homework, which is not surprising because it was the form of writing most used in the class. In addition, the instructions on the worksheet served as a source of confusion, with some students answering the worksheet questions based on the model article rather than their own experimentation.

Most of the errors present could be attributed to audience identification. For example, failing to properly label and describe figures could be because students assumed that readers knew the experimental setup. Likewise, context and rationale for an experiment would be obvious to an instructor, but is necessary nonetheless for the “knowledgeable peer” audience that the report was supposed to be written for. However, these areas all showed improvement in the final drafts.

It has been previously reported that novice writers in the biological sciences had difficulty with writing style, particularly the use of passive voice and self-reference (Stockton, 1994). However, relatively few students in this study showed substantial weakness in this area, with most students able to use passive voice effectively. Only 3 of 24 students showed appreciable problems with voice, and 2 of them solicited direct instruction on correcting their writing. In these sessions, I would read aloud a sentences or group of sentences the student had written, and ask them to rephrase it without using personal pronouns. After 3 or 4 examples, I asked the student to identify the problem sentences on their own and reword them for me. These one-on-one instruction sessions made previously opaque stylistic issues apparent and offered practice on remedying them.

Finally, a trend I noticed that I had not thought about when planning the intervention was an increased willingness of students to ask for assistance. At the beginning of the semester, I was told that previously the draft of the Materials and Methods and Results section had been voluntary, and that only 2-3 students in the class had turned in drafts. However, by making the drafts mandatory, every student received feedback. In addition, several students asked for comments on a second draft, so I posted office hours for revisions. 12 out of 24 students came to office hours to ask questions about their drafts or offered additional sections of their reports for feedback. It seems that early feedback on performance may either make the assignment seem more important to the student, make them aware of specific areas needing improvement, or make asking for assistance less intimidating. Given that the assignment was only worth 6% of the class grade overall, the amount of student-initiated interaction was impressive. References Curto, K., and Bayer, T. (2005). Writing and Speaking to Learn Biology: An Intersection of Critical Thinking and Communication Skills. Bioscene 31: 11-19. Mukhopadhyay, S. (2011) M435: Tissue Culture and Virology Laboratory, Kearney, NE, RLSimmonson Studios. Stockton, S. (1994). Students and Professionals Writing Biology: Disciplinary Work and Apprentice Storytellers. Language and Learning Across the Disciplines. 1: 79-104.

M435 – Writing Survey #1 Name:________________ Due 04/12/11 1. How many times have you written a paper based on your own data? (i.e. a

lab report) 2. Use the scale below to answer the following questions.

Very Confident/Good No Confidence/Poor

1 2 3 4 5 How do you perceive your ability to:

a. Organize the content of your paper?

1 2 3 4 5

b. Include the correct level of detail and types of information in your paper?

1 2 3 4 5

c. Generate meaningful and useful tables and figures of your data?

1 2 3 4 5

d. Write purposeful and coherent sentences and paragraphs?

1 2 3 4 5

e. Correctly use scientific terminology?

1 2 3 4 5

f. Select, understand and cite references?

1 2 3 4 5

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Sept. 1998, p. 7349–7356 Vol. 72, No. 9

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Binding of Sindbis Virus to Cell Surface Heparan SulfateANDREW P. BYRNES1 AND DIANE E. GRIFFIN1,2*

Departments of Molecular Microbiology and Immunology1 and Medicine and Neurology,2

Johns Hopkins University School of Hygiene and Public Health,Baltimore, Maryland 21205

Received 26 February 1998/Accepted 5 June 1998

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cellsurface receptors which allow infection of so many different species and cell types are still incompletelycharacterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate inthe binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and thisinteraction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experimentsshowed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However,Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate,indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross Rivervirus, was found to be independent of heparan sulfate.

The alphaviruses belong to a genus of enveloped RNA vi-ruses which can replicate in insects, birds, and mammals, in-cluding humans (60). They have a wide geographic distribu-tion and pose a serious threat to human health in certainregions, causing fever, rash, arthralgia, myalgia, and fatal en-cephalitis. In mammals, some alphaviruses have tropisms forspecific cell types such as muscle, neurons, and lymphaticcells. A better knowledge of the cellular receptors used byalphaviruses would have obvious implications for under-standing of the different cellular tropisms and pathogenesesof these viruses, as well as applications to the design of safelive-attenuated vaccines.

Alphavirus virions have a simple structure, with a singlestrand of positive-sense RNA enclosed in an icosahedral cap-sid, which is surrounded by a lipid envelope derived from thehost plasma membrane. The envelope contains two viral gly-coproteins, E1 and E2, which are organized in spikes. Duringthe initiation of infection, E2 is mainly responsible for bindingto cellular receptors. Following endocytosis, a low-pH-depen-dent rearrangement of the glycoproteins occurs, triggering themembrane fusion activity of E1 and allowing entry of thecapsid into the cytoplasm.

Alphaviruses cycle alternately between vertebrates and he-matophagous insects (usually mosquitoes), suggesting eitherthat virions bind to receptors that are highly conserved be-tween species or that the virus can use multiple receptors. Aprevious study has identified a role for the 67-kDa high-affinitylaminin receptor in binding of Sindbis virus (SV) to rodent andmonkey cells but not to avian cells (68). Another study usingVenezuelan equine encephalitis (VEE) virus identified a 32-kDa receptor in mosquito cells which also appears to be a lam-inin receptor (34). Other studies have identified unknown 74-and 110-kDa proteins as possible receptors for SV on mouseneuroblastoma cells (66) and a 63-kDa protein on chicken cells(69).

The normal in vivo role of glycosaminoglycans (GAGs) is tobind a diverse group of growth factors, chemokines, enzymes,and matrix components (20). In addition, however, these car-bohydrates are important in the cell surface binding of a num-ber of bacteria, parasites, and viruses (52). GAGs are un-branched polysaccharides present ubiquitously on cell surfacesand in the extracellular matrix and are usually found covalentlyattached to core proteins (proteoglycans) (31, 61). Some com-mon types of GAG include heparan sulfate (HS), chondroitinsulfate (CS), dermatan sulfate, and keratan sulfate. GAGs ac-quire a net negative charge through N and O sulfation, andGAG-binding domains of proteins are typically positivelycharged regions containing arginine and lysine. Importantly,GAGs are found in a wide variety of vertebrate and inverte-brate species, including insects (7).

The first virus found to bind HS was herpes simplex virus(HSV) (70), and since then a number of other herpesviruseshave been demonstrated to use HS as an initial receptor (43,45, 58). In addition, recent work has shown that HS is alsoinvolved in the binding of human immunodeficiency virus type1, foot-and-mouth disease virus, respiratory syncytial virus,dengue virus, and adeno-associated virus type 2 (9, 21, 32, 47,62). It should be noted, however, that additional receptorsbesides HS are involved in binding and entry of many, perhapsall, of these viruses.

There is suggestive evidence that GAGs may be involved inthe binding of alphaviruses to cells. Binding appears to involveelectrostatic interactions between ionizable groups on the virusand the cell membrane—it is highly dependent on the pH ofthe medium, and binding is reduced in medium of elevatedionic strength (15, 18, 34, 37, 38, 49). Studies have also shownthat polyanions, including sulfated polysaccharides such as hep-arin, can influence binding of alphaviruses. When present dur-ing viral absorption, polyanions reduce the number of plaquesformed in plaque assays (40), and pretreatment of certain cellswith heparin can increase binding of SV (63). A sulfated poly-saccharide contained in agar has been known for many years toinhibit the growth and decrease the plaque size of alphaviruses(4, 11, 57). Finally, the finding that treatment of cells with hep-arinase reduces the plaque-forming efficiency of SV (40) di-rectly suggests that SV might bind to HS.

* Corresponding author. Mailing address: Department of MolecularMicrobiology and Immunology, Johns Hopkins University School ofHygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205.Phone: (410) 955-3459. Fax: (410) 955-0105. E-mail: dgriffin@welchlink.welch.jhu.edu.

7349

MATERIALS AND METHODS

Chemicals and antibodies. The following were obtained from Sigma (St.Louis, Mo.): heparin (183 U/mg, from porcine intestinal mucosa), chondroitinsulfate A (CS-A) (bovine trachea), CS-B (porcine skin), CS-C (shark cartilage),dextran (molecular weight, 500,000), heparinase I (EC 4.2.2.7), and chondroiti-nase ABC (EC 4.2.2.4; affinity-purified). Dextran sulfate (17% S) and DEAE-dextran were obtained from Pharmacia (Piscataway, N.J.). The following wereobtained from Seikagaku America Inc. (Rockville, Md.): HS (bovine kidney; 5.0to 6.0% S), N-desulfated, N-acetylated heparin (,0.2% NS, .8.0% S); com-pletely desulfated N-acetylated heparin (,0.1% NS, ,1.5% S); completely de-sulfated N-sulfated heparin (.4.5% NS, 4.5 to 7.0% S).

Monoclonal antibodies were obtained as ascites from BALB/c mice. Thefollowing monoclonal antibodies were used: 202 immunoglobulin G3 (IgG3)against SV E2 epitope ab (42), R6 IgG2a against SV E2 epitope c (46), and thecontrol antibody 3E1, an IgG1 recognizing HSV (HB-8067, from the AmericanType Culture Collection [Manassas, Va.]). IgG was purified from ascites with thePierce T-Gel purification kit. Purified IgG was stored in frozen aliquots until use.Fab fragments were prepared by papain digestion with the Pierce ImmunopureFab preparation kit and were separated from Fc and undigested IgG with aprotein A-Sepharose column. Concentrations of antibody were determined byoptical densitometry at 280 nm.

Viruses and cells. Chinese hamster ovary (CHO-K1) cells (CCL-61) and theGAG-deficient CHO derivatives pgsA-745 (CRL-2242), pgsD-677 (CRL-2244),and pgsE-606 (CRL-2246) (2, 13, 33) were obtained from the American TypeCulture Collection and grown in Ham’s F-12 medium supplemented with 10%fetal calf serum and 50 mg of gentamicin per ml. BHK-21 cells were grown inDulbecco’s modified Eagle medium with the same supplements. SV strain Toto1101 (51) and Ross River virus (RRV) strain T48 (27) were grown and titered onBHK-21 cells.

For 35S-labeled viral stocks, BHK cells were infected for 1 h at a multiplicityof infection of approximately 2. Three hours later, cells were rinsed and themedium was replaced with Met–Cys-free Dulbecco’s modified Eagle mediumcontaining 1% fetal calf serum and 35 mCi of [35S]Met-Cys per ml. Supernatantfluid was collected at 24 h postinfection and clarified by centrifugation. A one-third volume of 40% polyethylene glycol 8000 in 2 M NaCl was added, and themixture was rocked overnight at 4°C. Virus was precipitated by centrifugation at18,000 3 g for 1 h and resuspended in a small volume of phosphate-bufferedsaline (PBS). Virus was applied to the top of a linear 15 to 40% (wt/vol)potassium tartrate gradient in PBS and centrifuged for 1.5 h at 190,000 3 g. Theviral band was collected and pelleted by centrifugation through a 15% sucrosecushion for 30 min at 240,000 3 g. Virus was resuspended in a small volume ofbinding buffer: PBS (pH 7.2) supplemented with 0.5 mM MgCl2, 0.5 mM CaCl2and 0.5% bovine serum albumin (BSA). Virus was stored in aliquots at 270°C.The counts per minute (cpm)/PFU ratio for SV Toto 1101 was 9.0 3 1024. ForRRV T48, the ratio was 3.8 3 1023.

Plaque assays. Virus was diluted in PBS supplemented with 0.5 mM MgCl2and 0.5 mM CaCl2. Polyanion inhibitors were added as noted in Results. Themedium was removed from confluent BHK monolayers in 35-mm wells, and 200ml of virus was added. Cells were infected for 1 h in a humidified 5% CO2atmosphere at 37°C, with occasional rocking. Cells were then overlaid with warmmodified Eagle medium containing 0.6% Bacto Agar (Difco, Detroit, Mich.) and1% fetal calf serum and lacking phenol red. Plaques were stained at 2 days withneutral red. When CHO cells (or their derivatives) were used for plaque assays,agarose was used instead of agar (to increase plaque diameter), and the overlaywas supplemented with nonessential amino acids.

Cell-binding assay. Cells were plated at 4 3 105 per well in 12-well plates andused the following day. Medium was removed from the cells, which were thenrinsed twice on ice with ice-cold binding buffer (PBS [pH 7.2], 0.5 mM MgCl2, 0.5mM CaCl2, 0.5% BSA). Approximately 104 cpm of 35S-labeled virus was addedto each well in 150 ml of binding buffer, and plates were rocked at 4°C for varyinglengths of time. Virus was removed and monolayers were rapidly rinsed twicewith ice-cold binding buffer. Cells were lysed in 1% sodium dodecyl sulfate, andcpm were assayed by liquid scintillation.

In some experiments, cell monolayers were pretreated with heparinase orchondroitinase. Enzyme incubations were performed in binding buffer at roomtemperature with constant shaking for 1 h. Monolayers were rinsed twice, andvirus binding was assayed as described above at 4°C.

Polyanion-binding assay. Because GAGs bound poorly to plastic plates, weadapted the method of Yang et al. (71) to allow covalent coupling of polysac-charides through their carboxyl groups. Ninety-six-well plates precoated withreactive hydrazide linkers (Corning Costar, Wilkes Barre, Pa.) were incubatedwith 25 mg of polysaccharide per well in 100 ml of 100 mM 1-ethyl-3-(3-dimeth-ylaminopropyl)carbodiimide and 50 mM borate, pH 5.2. Incubation was at roomtemperature overnight with constant shaking. Plates were rinsed three times with0.5 M sodium acetate, pH 4.0, and incubated for 30 min in this solution. Plateswere rinsed three times with PBS and blocked with PBS containing 0.5% BSA for30 min. Virus (5 3 103 to 1 3 104 cpm) in 100 ml of PBS (pH 7.0) with 0.5% BSAwas added to each well. All incubations were performed at room temperaturewith constant shaking. After 2 h, 50 ml from each well was collected and countedby liquid scintillation. The amount bound to the plate was calculated by sub-tracting the resulting cpm from the cpm in an uncoated well. When antibody was

used to block binding, virus and antibody were mixed for 30 min at roomtemperature before being added to the 96-well plate.

Heparin-Sepharose chromatography. Prepacked 1-ml HiTrap heparin-Seph-arose columns (Pharmacia) were equilibrated at room temperature with 10 ml of100 mM NaCl–5 mM phosphate (pH 7.5)–0.5% BSA at a flow rate of 1 ml/min.Approximately 105 cpm of 35S-labeled virus was added in 1 ml of the same buffer,followed by 4 ml of buffer. The virus was eluted with a 40-ml linear gradient from100 to 500 mM NaCl containing 5 mM phosphate (pH 7.5) and 0.5% BSA.One-milliliter fractions were collected and counted by liquid scintillation. Anyremaining virus was removed from the column by using 0.5% sodium dodecylsulfate. The NaCl concentration of each fraction was determined by measuringthe conductivity of a 50-fold-diluted aliquot.

Statistical analysis. All results are expressed as means 6 standard deviations.Error bars in graphs represent standard deviations. Unless otherwise noted,results were tested for significance by analysis of variance (ANOVA), followed bythe Tukey test to determine differences among groups. Results having P valuesof ,0.05 were considered significant.

RESULTS

Inhibition of plaque formation by GAGs. It has previouslybeen reported that certain polyanions, including some GAGs,decrease the number of SV plaques when present in the me-dium during the binding step of plaque assays (40). This mightbe interpreted as a type of competition experiment; an excessof polyanions in the medium could be preventing binding ofthe virus to a cell surface polyanion.

The major GAGs found on most cells are HS and CS. Weexamined this blocking phenomenon further in plaque assayson BHK cells by using heparin (a highly sulfated version of HS)and the three forms of CS found on the cell surface: CS-A,CS-B (also known as dermatan sulfate), and CS-C. Heparinand dextran sulfate (an artificial, highly sulfated polysaccha-ride) inhibited plaque formation to similar degrees (Fig. 1A).

FIG. 1. Inhibitory effects of polysaccharides on plaque formation by SV. SVstrain Toto 1101 was used throughout this study. (A) SV was mixed with variousGAGs or the highly sulfated polysaccharide dextran sulfate and incubated onmonolayers of BHK cells for 1 h at 37°C before being overlaid with agar. (B) Therequirement for high sulfation was shown by the inability of HS or various formsof desulfated heparin to inhibit SV plaque formation. Abbreviations: CDSNS,completely desulfated, N sulfated; CDSNAc, completely desulfated, N acety-lated; NDSNAc, N desulfated, N acetylated. Error bars are omitted for clarity ofpresentation. Each point is the mean of three or more measurements. p, P of,0.05 versus no inhibitor.

7350 BYRNES AND GRIFFIN J. VIROL.

M435 – Writing Sample #1 Name:__________________ Due 04/05/11 The J. of Virology article, “Binding of Sindbis Virus to Cell Surface Heparin Sulfate” can be used as a model for your writing. Important parts of the article have been marked or highlighted to point out elements of writing structure and organization corresponding to points in the “Unknown Virus Lab Report Guidelines”. Read through the results subsection that has been marked-up. Then, read through another subsection of the results and identify the same aspects of structure and organization. Your results section can be broken down into subsections based on each of the experiments you performed in identifying your unknown. For each subsection, you will want to address certain points—the same ones pointed out in the model article. You can use the questions below to guide your writing.

1. What aspect of viral biology is the experiment testing or what are you trying to learn about the virus? Formulate a one sentence goal or hypothesis that explains what is being tested.

2. How are you testing this particular aspect of viral biology? (What is the experiment?)

3. What are the variables and controls and what will they tell you about either the experiment or the virus?

4. What were the results? Design a figure that shows your results, and write a few sentences pointing out the most important aspects of the results.

M435 – Lab Report Draft Assessment Rubric Student Name:________________ Materials and Methods:

Performance Parameter Good Average Needs

Work Writing: Subsection Headings Complete Sentences Passive tense, non-narrative Paragraph form Brevity Content: Includes volumes, reagent concentrations and sources where appropriate Complete steps, nothing missing A peer virologist could replicate experiment Overall Impression

Comments: Results:

Performance Parameter Good Average Needs

Work Writing: Subsection Headings Complete Sentences Passive tense, non-narrative Paragraph form Brevity Content: Rationale for experiment Important Controls and Variables Major Observation/Result clearly stated in text Tables/Figures Referenced in text No Methods; No Interpretations Overall Impression

Comments: Figures and Tables:

Performance Parameter Good Average Needs

Work Figure Numbered and Titled Figure described in words below figure Figure description separated from body of results Figure legend Labels present and complete Accurately represents data Neatly and logically presents data (not confusing) No raw data Overall Impression

Comments:

M435 – Writing Survey #2 Name:________________ Due 04/28/11 **Your answers to these comments have no impact on your grade. They are used to evaluate the methods of instruction used on this assignment. Please answer honestly.** 1. Did you use the worksheets to aid you in your writing? 2. If you used the worksheets, using the scale below, rate their usefulness.

Very Helpful Not Helpful 1 2 3 4 5

3. What aspects of the worksheet were most helpful? Please be as specific as possible. 4. What comments on your draft were most helpful? Please explain.

(Continued on next page)

5. After writing your final paper, use the scale below to answer the following questions.

Very Confident/Good No Confidence/Poor

1 2 3 4 5 How do you perceive your ability to:

a. Organize the content of your paper?

1 2 3 4 5

b. Include the correct level of detail and types of information in your

paper?

1 2 3 4 5 c. Generate meaningful and useful tables and figures of your data?

1 2 3 4 5 d. Write purposeful and coherent sentences and paragraphs?

1 2 3 4 5 e. Correctly use scientific terminology?

1 2 3 4 5

f. Select, understand and cite references?

1 2 3 4 5

6. What changed most substantially about your writing between the first and

final drafts?

M435: Virology and Tissue Culture LaboratorySpring 2011

Tuesday and Thursday 1:25-4:25Jordan Hall 022

Instructor: Tuli MukhopadhyayAIs: Joyce Patrick and Yahong Wen

Class overview. The objective of M435 is to introduce students to basic concepts and experimental techniques used in a virology laboratory. After taking the class, you should understand the theory and have practical experi-ence to successfully work in a virology research laboratory. You should have taken or are currently taking the virology lecture course, M430.The class is divided into three modules: cell culture, fundamental virology, and identification and characterization of an unknown virus sample. Modules I and II will be covered before spring break and Module III will be covered after spring break.Module I: Cell culture. The objective of this module is to learn fundamental cell culture techniques including sterile technique, cell maintenance, cell-splitting, and freezing cells.Module II: Fundamental virology. The objective of this module is to learn standard assays used in virology studies such as virus amplification, plaque assays, host-range studies, and ELISAs. In addition to learning the technical details, we will focus on how to interpret, analyze, and evaluate experimental results.Module III: Identification and characterization of unknown viruses. The objective of this module is to identify and characterize an unknown virus sample based on the techniques and analysis methods learned in the first half of the semester. You will summarize this module by writing a journal article that emphasizes the significance and results from your work.Textbook. The lab manual for this course is “M435 Tissue Culture and Virology Lab Manual, Spring 2011, edi-tion 3” by Tuli Mukhopadhyay and published by RLSimonson Studios.Office hours. We strongly encourage individuals to contact either the AIs or the instructor is any aspect of the lab is not clear. As the semester progresses, you will build upon topics you learned earlier in the semester. We are happy to explain or discuss both theoretical and experimental aspects of the labs we perform in class. Office hours are by appointment.

Tuli Mukhopadhyay sumukhop@indiana.edu Office: SH 220CJoyce Patrick jopatric@umail.iu.edu Office: SH 406Yahong Wen yahwen@imail.iu.edu Office: SH 209

Assignments and grades. You semester grade is based on five different components, each detailed below. The assignments emphasize the main topics and the take-home lessons I would like you to retain. Experiments, both the theoretical and experimental aspects, build upon concepts from previous experiments. The purpose of lab worksheets is to make sure the main points of each laboratory experiment are understood. Furthermore, the homework assignments are a means to make sure you to be able to relate different concepts to each other. Please see your AI or me if you are having difficulty with the lab worksheets or homework assignments since these are the basis for future experiments as well as the exams. No assignments will be accepted late unless you have an excused absence.

Experiment worksheets. Each experiment will have an accompanying worksheet. These worksheets are meant to be filled in as you are performing the experiment and guide you as you perform the experiment. These worksheets also provide a place to record data, do calculations, and analyze your results. During Module III the worksheets will guide you in identifying your unknown virus. Please see the syllabus and calendar for when worksheets are due.Homework. The homework assignments are a combination of concepts and principles, data analysis, and are based on the lab and pre-lab lectures (see the syllabus and calendar for when worksheets are due). The homework assignments are meant to be a check-point to ensure you understand the main concepts of what is being taught.Exams. Two exams are given in this course. The first exam is before spring break. The exam is short answer and covers the principles and concepts taught during Modules I and II. Thesecond exam is at the end of the semester and is a lab practical. Here you are shown actual results and asked to interpret, analyze, and draw conclusions. Lab worksheets, pre-lab lectures, and homework assignments are your best guide for preparing for these exams. If you have university documentation showing you need extra time for an exam, please let me know within the first two weeks of class.Unknown virus write-up. For Module III, Identification and Characterization of an Unknown virus, you will sum-marize your results as a scientific paper. You will detail how you determined the identity of your unknown virus and its properties. Results from each experiment will be summarized as a figure or table, and an appropriate intro-duction and discussion should be included. Guidelines for how to write this paper are at the end of the lab manual and we will discuss this more during the second half of the semester. To help you learn about scientific writing, you will be required to turn in at least one “Results” section and its corresponding “Materials and Methods” sec-tion before turning in the final unknown lab write-up.Lab performance. There are many times during the semester that you will need to come in during non-lab hours to finish an experiment or record results. Experiments can be complicated, require reagent preparation beforehand, and require care since you will be working with live virus. Your lab performance grade will be based on ability to maintain cell lines, preparing for an experiment, and returning to finish an experiment. This will be determined by both your AI and me.The five different components contribute to your final grade as listed below:Experiment worksheets 500 points 25 points/each 20 totalHomeworks 400 points 50 points/each 8 totalExams 400 points 200 points/each 2 totalUnknown virus write-up 100 pointsLab performance 100 points 1500 points total

% Score Range Points Letter Grade92-100 1380-1500 A90-91 1350-1379 A-88-89 1320-1349 B+82-87 1230-1319 B80-81 1200-1229 B-78-79 1170-1199 C+72-77 1080-1169 C70-71 1050-1079 C-68-69 1020-1049 D+62-67 930-1019 D60-61 900-929 D-< 59 <899 F

Laboratory experiments. An experiment may take 1-2 weeks to finish. Many labs require a few days of prepa-ration (usually getting your cells ready), a day to do the experiment, and another day to obtain and analyze the results. To help you keep track of the experiments a detailed schedule has been made. This is in the lab manual and on Oncourse as a .pdf file. The schedule is only a guide and should be used as the first step in planning your experiments. It is your responsibility to have your cells ready on the day needed and to come into lab and record your results when necessary.Students will work in groups of two. Each group will work in their own hood and be responsible for cleaning the hood, discarding trash and bringing pipettes and biohazard waste to front of the room at the end of each lab pe-riod. Each group will receive some initial supplies the first day which they are responsible for maintaining. Both partners must attend class and participate in the lab. If you have to miss a class (illness, job interview), please let your lab partner and myself know as soon as possible.Laboratory safety. We will be working mainly with Sindbis virus and BHK (Baby hamster kidney) cells but we will do some work with other viruses and cell lines. If you are under a physician’s care for an immune related condition, please consult with your physician about taking the class.Incomplete course policy. As indicated by Indiana University Policy “A grade of I (Incomplete) may be given only when the work of the course is substantially completed and when the student’s work is of passing quality.”Academic integrity policy. Academic integrity is important for the fair assessment of each student’s perfor-mance. Each student is expected to adhere to the academic integrity policies and regulations outlined by the Indiana University Code of Student Rights, Responsibilities, and Conduct (http://campuslife.indiana.edu/Code). Cases where academic integrity is clearly in question will be pursued and individuals involved in these incidents should expect a failing grade for the course and further prosecution through the IU academic integrity policy. Common sense should be used in abiding by these regulations, and any ambiguities should be checked out with the AI and/or the instructor. Examples of academic integrity violations include but are not limited to: 1) submit-ting another student’s work as your own, 2) giving or receiving unauthorized assistance, and 3) plagiarism.Plagiarism. Although you will complete experiments in pairs, each person in the group is expected to complete the worksheets, homework assignments, write-up, and exams individually. Our class will follow the procedure for cheating, fabrication, and plagiarism as taken from the Instructional Support Services Webpage:“Plagiarism constitutes using others’ ideas, words or images without properly giving credit to those sources. If you turn in any work with your name affixed to it, I assume that work is your own and that all sources are indi-cated and documented in the text (with quotations and/or citations). I will respond to acts of academic misconduct according to university policy concerning plagiarism; sanctions for plagiarism can include a grade of F for the assignment in question and/or for the course and must include a report to the Dean of Students Office.”

Wk Day Date Experiments(scheduled lab days)

Follow-up(non-lab days) Assignments due

MODULE I: INTRODUCTION TO CELLS

1 T 1.11

• Experiment #1: Introduction to cells: im-portanct of sterile technique

• Check in & get supplies• Discuss lab safety and biosafety• Informational sheet

R 1.13• Continue/Finish Experiment #1• Experiment #2: Introduction to cells: visu-

alizing different cell types and determin-ing cell viability and concentration

• Worksheet Experiment #2 due

2 T 1.18• Finish Experiment #1• Experiment #3: Introduction to cells:

learning to passage and maintain cells• Worksheet Experiment #1 due

W 1.19 • Continue Experiment #3

R 1.20 • Continue Experiment #3 • Homework #1 due

WE 1.22/1.23

• Continue Experiment #3

3 T 1.25 • Continue Experiment #3 • Worksheet Experiment #3 due

R 1.27 • Experiment #4: Introduction to cells: freezing and storing cells • Homework #2 due

MODULE II: INTRODUCTION TO VIRUSES

4 T 2.1• Continue Experiment #4• Experiment #5: Introduction to viruses:

determining infectivity by plaque assay

R 2.3 • Finish Experiment #4• Finish Experiment #5

• Worksheet Experiment #4 due• Worksheet Experiment #5 due

5 T 2.8 • Experiment #6: Introduction to viruses: propagating virus samples

W 2.9 • Continue Experiment #6

R 2.10 • Continue Experiment #6 • Homework #3 due

F 2.11 • Finish Experiment #6

6 T 2.15

• Experiment #7: Introduction to viruses: determining how MOI influences infectiv-ity

• Experiment #8: Introduction to viruses: determining which hosts are susceptible to viral infection

• Worksheet Experiment #6 due

W 2.16• Continue

Experiment #7 and #8

R 2.17• Finish Experiment #7• Finish Experiment #8• Experiment #9: Introduction to viruses:

determining viral growth kinetics

• Worksheet Experiment #7 due• Worksheet Experiment #8 due• Homework #4 due

Wk Day Date Experiments(scheduled lab days)

Follow-up(non-lab days) Assignments due

F 2.18 • Continue Experiment #9

7 T 2.22 • Continue Experiment #9R 2.24 • Finish Experiment #9

F 2.25 • Worksheet Experiment #9 due• Homework #4 due

8 T 3.1 • Experiment #10: Characterization of viruses: ELISA assay

• Worksheet Experiment #10 due

R 3.3• Experiment #11: Characterization of

viruses: pH and lipid sensitivity• Experiment #12: Characterization of

viruses: hemagglutination assay

• Worksheet Experiment #11 due

F 3.4 • Finish Experiment #11 • Homework #5 due

9 T 3.8 • Experiment #13: Characterization of viruses: electron microscopy

• Worksheet Experiment #11 due

• Worksheet Experiment #13 due

R 3.10 • EXAM IT 3.15 Spring BreakR 3.17 Spring Break

MODULE III: CHARACTERIZATION TO VIRUSES

10 T 3.22• Experiment #14: Identification of un-

known virus• Determine host-range of unknown virus

• As needed for all experiments during rest of semester

• Turn in worksheets as you fin-ish experiments

11 T 3.29 • Characterization of pH and lipid sensitiv-ity of unknown virus

R 3.31 • Start amplifying unknown virusF 4.1 • Homework #6 due

12 T 4.5 • Determination of nucleic acid type in unknown virus

F 4.8 • Homework #7 due

13 T 4.12 • Determine infectivity of amplified un-known virus by plaque assay

• Methods and results for at least one experiment

F 4.15 • Homework #8 due14 T 4.19 • Repeat experiments if necessary

R 4.21• Last day for experiments• Clean-up• Check-out

F 4.22 • Last day to turn in worksheets #14-#20

15 T 4.26 • EXAM II, Lab practical

R 4.28 • Write-up for Experiment #14 due

UNKNOWN VIRUS LAB REPORT GUIDELINES

ABSTRACT (10 points): The abstract should be a one paragraph, concise summary of the unknown virus lab. It should address the following: -goal and rationale for the project -a very brief description of methodology used -summary of the major results and conclusions

INTRODUCTION (10 points)The introduction should provide the reader the information needed to understand the project. It should de-scribe the following: -experimental goals/hypotheses -experimental rationale; why bother doing the experiment? -significance to the field

MATERIAL AND METHODS (10 points)The material and methods section is a concise and accurate description of the procedures used in your report. A virologist should be able to duplicate your experiment if given only this section. Please look at an article from Journal of Virology for an example of what we will be looking for. This section should include: -titled subsections -brief but complete description of methods written in paragraph form

We do not want to see: -Results -Stepwise protocols -Flowcharts

RESULTS (30 points)In the results section, present the data that the reader will need later for your interpretations (which will be found in discussion). Whenever possible, present data in a table or graph. For example, the results of the growth curve experiment should be a graph, not a picture of each plaque assay plate. If you have to repeat certain experiments, only write about the final one that worked. This section should include: - titled subsections

- brief context and rationale for each experiment. For example, “In order to determine X, we did Y.” De-tails of how you did Y will be found in materials and methods.

- how do you control for your experiment- At the end include a statement of major data trend or observation

- DO NOT include any statements of data interpretations or conclusions - reference tables and figures in the text, not at the end of the report - tables and figures should have legend titles and descriptions (see Journal of Virology article) - tables and figures should be properly labeled and presented neatly and logically

Identification of an Unknown Virus 91