Identification of an Unknown Virus

1
Identification of an Unknown Virus
Aaron Weston, Joe Julian, Jess Savino, Steve
Berman
Abstract
Methods
Results

• To determine if unknown nucleic acid was RNA or
  DNA
• Bials Reaction
• In two microcentrifuge tubes, 10 µL of the
  unknown nucleic acid was pipetted
• Into tube 1, 100 µL of 6 alcoholic orcinol was
  pipetted
• Into tube 2, 40 µL of 6 and 60 µL of orcinol
  acid reagent were pipetted
• Both tubes were placed in 94C thermal cycler for
  20 minutes then allowed to cool
• Absorbance for each sample was then measured at
  660 nm
• Process was repeated two more times
• Dische Reaction
• In two microcentrifuge tubes, 10 µL of the
  unknown nucleic acid was pipetted
• Into tube 1, 90 µL of distilled water was
  pipetted
• Into tube two, 30 µL of distilled water and 60 µL
  of diphenylalanine was pipetted
• Both tubes were placed in 94C thermal cycler for
  10 minutes then allowed to cool
• Absorbance for each sample was then measured at
  595 nm
• Process was repeated two more times
• To determine if unknown acid was single- or
  double-stranded
• Digestion of unknown nucleic acid with S1
  nuclease

A virus infected over 30 children in one
week in Virulent, Pennsylvania. The possible
choices for the virus have been narrowed down to
orthomyxovirus, reovirus, parvovirus, and
papillomavirus. By using Bials reaction, Dische
reaction, and gel electrophoresis, the structure
and composition of the unknown virus was
identified and compared to the background
information obtained on the 4 viruses. The tests
showed the unknown virus was single-stranded and
composed of RNA. These properties matched those
of the orthomyxovirus. The orthomyxovirus was
concluded to be the disease-causing virus in this
case.
Discussion Conclusions
As Table 1 shows, the absorbance value for
the unknown nucleic acid in the Bials reaction
corresponded to a green color. At the same time,
the absorbance value for the unknown nucleic acid
in the Dische reaction shows that no reaction
took place because of its clear appearance. The
positive test for the Bials reaction indicates
that the unknown nucleic acid is RNA. As
Figure 2 shows, the electrophoresis of the
unknown nucleic acid shows a clear band about
one-third down the gel. However, in the presence
of the S1 nuclease, the electrophoresis is almost
invisible. This is because the unknown nucleic
acid was cleaved by the nuclease, meaning it was
single-stranded in its structure. The
results of the experiment show that the unknown
nucleic acid is RNA and single-stranded in its
nature. Based on the background research done on
the four possible choices of the nucleic, the
unknown nucleic acid sample was deduced to be an
orthomyxovirus.
Introduction
A mysterious virus struck the town of
Virulent, Pennsylvania, infecting over 30
children within a week. Some of the symptoms
displayed are nausea, vomiting, fever, and muscle
aches. The possible choices for the cause of the
virus have been narrowed down an orthovirus, a
reovirus, a parvovrius, or a papillomavirus
(Tobin-Janzen, 2004). A sample of the unknown
virus was to be analyzed until a specific virus
could be pinpointed. An unknown sample of
genetic material can be identified by various
tests, such as the Bials reaction and Dische
reaction. The Bials reaction is a test for
ribose. RNA can be hydrolyzed into a mix of
purines, ribose sugars, and pyramidine
nucleotides when it is mixed with a concentrated
acid (Tobin-Janzen, 2004). The ribose sugars
form intermediates via dehydration and react with
orcinol when ferric ions are present
(Tobin-Janzen, 2004). This gives the mixture a
blue or green color (Tobin-Janzen, 2004). The
Dische reaction is a test for deoxyriboses.
Deoxyribose sugars that are generated from the
hydroxylation of DNA form aldehydes that are blue
in color when reacted with diphenylamine
(Tobin-Janzen, 2004). Both tests use visual
color changes to determine whether a reaction a
took place. Gel electrophoresis can be used
to determine the structure of the unknown genetic
sample. Double-stranded and single-stranded
nucleic acid often react differently with various
enzymes. S1 Nuclease can cleave internal
phosphodiester bonds, which means it will cleave
single-stranded nucleic acids, but not
double-stranded ones (Tobin-Janzen, 2004). This
is because the double-stranded nucleic acids do
not expose their individual nucleotides, so the
S1 Nuclease cannot cleave individual base
sequences at normal concentrations. The
structures of the 4 viruses are different, so the
combination of results from the aforementioned
tests should provide sufficient information to
identify the virus. The orthomyxovirus is RNA
and is singled-stranded in its structure (Fix,
Orthomyxoviruses, 1997). The reovirus is also
RNA, but is double-stranded (Zhang, et al, 2003).
The papillomavirus is DNA and is double-stranded
and circular in its structure (Fix,
Papovaviruses, 1997). The parvovirus is DNA and
is single-stranded (Sander, 1995). The results
of the tests should provide enough information to
identify the unknown virus.
Table 1. The absorbance values for the unknown
sample of genetic material after running the
Bials and Dische reactions.
References
Department of Biology. The University of York.
Structure and function of human papillomavirus
proteins. 2004. http//www.york.ac.uk/depts/bio
l/units/cru/hpv.htm. Accessed 26, Sept.
2004. Fix, Douglas F. Southern Illiniois
University-Carbondale. Orthomyxoviruses. 1997.
http//www.cehs.siu.edu/fix/medmicro/ortho.htm
. Accessed 26, Sept. 2004. Fix, Douglas F.
Southern Illiniois University-Carbondale.
Papovaviruses. 1997. http//www.cehs.siu.ed
u/fix/medmicro/papov.htm. Accessed 26, Sept.
2004. Reinish, K.M., et al. Cornell High Energy
Synchrotron Source. Structure of Reovirus Core
Determined Via X-ray Crystallography. 2004.
http//www.chess.cornell.edu/Research/Highli
ghts/ReovirusCore.htm. Accessed 26, Sept.
2004. Sander, D. University of Tulane. The Big
Picture Book of Viruses Parvoviridae. 1995.
http//www.tulane.edu/dmsander/Big_Virology/BVD
NAparvo.html. Accessed 26, Sept. 2004. Stannard,
Linda M. University of Cape Town. Influenza
Virus. 1995. http//web.uct.ac.za/depts/mmi
/stannard/fluvirus.html. Accessed 26, Sept.
2004. Wadsworth Center. Wadsworth Center NYU
State Dept. of Health. Human Parvovirus is
Erythema Infectiosm (Fifth Disease). 2003.
http//www.wadsworth.org//databank/b19virus.htm.
Accessed 26, Sept. 2004. Tobin-Janzen, T.
Susquehanna University. The Case of the Virulent
Virus. 2004. http//comenius.susqu.edu/BI/2
01/01/Workshop20Genetics20Folder/virulentvirus.h
tml. Accessed 1, Sept. 2004. Zhang, X., et
al. PubMed. Reovirus polymerase lambda 3
localized by cryo-electron microscopy
of virions at a resolution of 7.6 A. 2003.
http//www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd
Retrievedb pubmeddoptAbstractlist_uids
14608373. Accessed 26, Sept. 2004.
Figure 2. The results of gel electrophoresis of
the unknown sample of genetic material with and
without S1 nuclease. The effects of the nuclease
on known samples of single-stranded and
double-stranded RNA are shown for comparison.