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October 18, 2012
VIRUS AND FORENSICS
MANISH SURVE, PRADIP HIRAPURE
Government Institute of Forensic Science, Mumbai
What is a Virus?
A virus is a small infectious agent that can replicate only inside the living cells of an organism.
Viruses can infect all types of organisms, from animals and plants to bacteria and archaea.
Viruses are constituted of two or three parts: the genetic material made from
either DNA or RNA, long molecules that carry genetic information; a protein coat that protects
these genes; and in some cases an envelope of lipids that surrounds the protein coat when they
are outside a cell. Some viruses, like HIV, have in addition an external envelope derived
from the plasma membrane of the host cell from which it came.
Forensic virology is the study of viruses in a forensic context.
Viruses can cause death, either as a natural process or as a criminal act (e.g. bioterrorism). These
viruses can be natural or engineered.
Forensic virology can be used to determine the type and strain of virus, and the source of an
engineered virus.
The phrase ‘trace-back investigation’ refers to the process of tracing a virus back to its source in
order to find out where an infection has come from.
Structure of Virus:
The capsid and entire viral structure can be of four main types:
1. Helical
There is a capsomer coiled around a central axis to form a helical structure. This is a common
structure seen in single stranded RNA viruses. Tobacco mosaic virus is a helical virus.
2. Icosahedral
These are near-spherical and this shape is adopted because the coat forms a closed shell.
Rota virus has twelve capsomers and appear spherical.
3. Envelope
The virus is covered with a lipid membrane in a modified form of one of the cell membranes.
The outer membrane is from the infected host cell and internal membranes from nuclear
membrane or endoplasmic reticulum forming a lipid bilayer known as a viral envelope. This
membrane is studded with proteins or receptors.
4. Complex
There is a capsid that is neither purely helical, nor purely icosahedral. There may be extra
features like protein tails or a complex outer wall. Bacteriophages are examples of this type of
viral structure.
MANISH DEEPAK SURVE, MSc-I, 20 Page 1

October 18, 2012
An array of viruses. (a) The helical virus of rabies. (b) The segmented helical virus of influenza.
(c) A bacteriophage with an icosahedral
herpes simplex virus. (e) The unenveloped polio virus.
(f) The icosahedral human immunodeficiency virus with spikes on its envelope.
How does virus function in a host cell?
MANISH DEEPAK SURVE
head and helical tail. (d) An enveloped icosahedral
SURVE, MSc-I, 20
Viruses lie around our environment all of
the time just waiting for a host cell to come
along. They can enter us through the nose,
mouth or breaks in the skin
find a host cell to infect. For example, cold
and flu viruses will attack cells
respiratory or digestive tracts. The human
immunodeficiency virus (HIV), which
causes AIDS, attacks the T
immune system.
Regardless of the type of host cell, all
viruses follow the same basic steps in what
is known as the lytic cycle (see figure):
once inside, they
that line the
T-cells of the
a. A virus particle attaches to a host
cell.
b. The particle releases its genetic
instructions into the host cell.
c. The injected genetic material recruits
the host cell's enzymes.
d. The enzymes make parts for more
new virus particles.
e. The new particles assemble the parts
into new viruses.
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October 18, 2012
f. The new particles break free from
the host cell.
Virus as Bioweapon:
Biological agents are likely to be used by terrorists as weapons because:
• They are capable of damaging populations, economies, and food supplies
• They can be directed at a small group of people or an entire population
• They can be used to attack people, economies and food supplies
• They cause fear, panic and social disruption
• Most of them are obtained from nature
• Certain agents are inexpensive to make
• They are easily made by relatively unsophisticated methods
• They are invisible to the senses
• Their effects may be delayed
Various delivery methods are possible:
· aerosol
· envelope or package
· food or water contamination
Viruses can cause death or ill effects in the body, either as a natural process or as a criminal act
(e.g. bioterrorism). These viruses can be natural or engineered.
Viral agents were first used as weapons by British General Jeffrey Amherst in 1763 against the
Native Americans during the French and Indian War. As a biological weapon, viral agents would
most likely be distributed in aerosolized form. Unlike bacteria, antibiotic treatments are not
effective therapies against viral infections. Inside a host, viruses may remain dormant for long
periods before reviving to infect other hosts.
Most common diseases caused by Virus used as bioweapon:
1. Small pox:
a. Infectious agent: Variola virus - Orthopox virus
b. Declared eradicated in 1980, but stockpiles may exist
c. Not naturally acquired. It can be disseminated as and inhaled as an aerosol
d. Symptoms: Fever, muscular rigidity, headaches, and vomiting. Severe cases
experience prostration and hemorrhage into skin and mucous membranes
e. Rash appears after about three weeks; progresses from macules (initial skin lesions) to
papules to pustular vesicles, to scabs
f. Transmission: may occur person to person by respiratory droplets or skin inoculation.
Highly contagious when rash appears
g. Incubation period: 10 to 12 days
h. Mortality: less than 1% in the minor form and 20 to50 % in the major form
i. Treatment: supportive
j. Prevention: vaccine
2. VIRAL HEMORRHAGIC FEVERS:
a. These are highly infectious viral illnesses caused by the Filoviruses (Ebola and
Marburg), Arenaviruses (Lassa fever), Bunyaviruses (Congo hemorragic fever and
Hantaviral disease), and Flaviviruses

October 18, 2012
b. Symptoms: vary from one type to the next. They include: sudden onset of fever, muscle
aches, headache, followed by vomiting, diarrhea, and rash and internal bleeding
c. Complications: In severe forms, multiorgan failure occurs, primarily due to hemorrhagic
and pulmonary complications
d. Mode of transmission: handling infected wild animals, but may be used as an aerosol
bioterrorist weapon
e. Incubation period: 2 to 21 days
f. Reservoir: gorillas and chimpanzees
g. Transmission: some may be spread person to person by contact with body secretions
h. Mortality: Ebola rates have reached 90% but vary
i. Treatment: supportive
j. Prevention: Avoid contact with infected monkeys or other animal hosts.
List of some viruses can be used as bio weapons:
• EBOLA
• LASSA FEVER
• INFLUENZA
• VIRAL HEPATITIS
• VIRAL HEMORRHAGIC FEVERS
• HIV etc
Techniques used for Detection and Investigation:
A. PCR
What is Polymerase Chain Reaction?
It is a fast and inexpensive technique used to amplify small and targeted segments of DNA to
produce million of copies, sometimes called "molecular photocopying" of a specific gene
fragment.
PCR Steps:
PCR is a three-step process which is repeated in several cycles. The three steps are:
1. Denaturation step: This step consists of heating the reaction to 90–95 °C. for 1 min. It causes
DNA separation by disrupting the hydrogen bonds between complementary bases, yielding
single strands of DNA.
2. Annealing step: The reaction temperature is lowered to 50–65 °C allowing hybridization of the
primers to the single-stranded DNA template. It require 1-2 min
3. Extension/Elongation step: At this step, the Taq polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding dNTPs that are complementary to the
template in 5' to 3' direction.
This process is repeated as many as 30 or 40 times, leading to more than one billion exact copies
of the original DNA segment. The entire cycling process of PCR is automated and can be
completed in just a few hours using a machine called a thermal cycler.

October 18, 2012
B. Random Amplified Polymorphic DNA (RAPD):
RAPD is based on the amplification of genomic DNA with single primers of arbitrary nucleotide
Sequence. These primers detect polymorphisms in the absence of specific nucleotide sequence
Information and the polymorphisms function as genetic markers and can be used to construct
genetic maps. Since most of the RAPD markers are dominant, it is not possible to distinguish
whether the amplified DNA segment is heterozygous (two different copies) or homozygous (two
identical copies) at a particular locus. In rare cases, co-dominant RAPD markers, observed as
different-sized DNA segments amplified from the same locus, may be detected.
The basic technique of RAPD involves (i) extraction of highly pure DNA, (ii) addition of single
random (arbitrary) primer, (iii) polymerase chain reaction (PCR), (iv) separation of fragments by
gel electrophoresis, (v) visualization of RAPD-PCR fragments after ethidium bromide staining
under UV light and (vi) determination of fragment size comparing with known molecular marker
with the help of gel analysis software. A diagrammatic presentation of these steps is given in
above Figure. It is important to note that RAPD technique requires maintaining strictly
consistent reaction conditions in order to achieve reproducible profiles. In practice, band profiles
can be difficult to reproduce between (and even within) laboratories, if personnel, equipment or
conditions are changed. Despite these limitations, the enormous attraction of this technique is
that there is no requirement for DNA probes or sequence information for primer design. The
procedure involves no blotting or hybridizing steps. Another advantage is the requirement for
only small amounts of DNA (10-100 ng per reaction).The RAPD markers have been used for
detecting genomic variations within and between individuals species. Genetic diversity was
evaluated by RAPD markers and RAPD has been used for estimation of microbial genetic
diversity.
C. T-RFLP (terminal restriction fragments length polymorphism):
Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one fingerprinting
methodology that can incorporate automated genotyping systems for laser detection
offluorescently-labelled DNA fragments. The T-RFLP analysis technique involves the
amplification of a gene of interest using fluorescent primers, followed by restriction
endonuclease digestion, and automated analysis of the end or terminal restriction fragments.
Because the 16S rRNA genes from all of the bacteria in the soil community are amplified, the
resulting PCR products are gene copies of a similar length but with different internal sequences.
The PCR products are cleaved with a restriction enzyme that recognizes and cleaves DNA at
particular sequences. In the variable regions of the 16S gene, restriction sites occur in different
places resulting in different length fragments. The more diverse the bacterial community in a
sample, the greater the range of resulting fragments. The end, orterminal fragments are analyzed
and the size and frequency of each fragment assessed to produce a profile or ‘fingerprint’ of the
whole bacterial community in a soil sample. The T-RFLP method was originally developed by
Avaniss-Aghajani et al. (1994) to identify Mycobacteria, but its potential to analyze variation
between genes from a mixture of bacteria was first shown by Liu et al. Since then, T-RFLP
analysis has been used in numerous studies to look at bacterial, archaeal and eukaryotic
populations in many different substrates, and has been identified as a reproducible and accurate

October 18, 2012
tool for community fingerprinting play a importance role in forensic investigation as a microbial
forensic tool.
SSCP (single stranded conformational polymorphism)
Increasingly in the genomics field, researchers are requiring screening and sequence variation
detection tools for large numbers of samples. SSCP analysis detects sequence variations (single-point
mutations and other small-scale changes) through electrophoretic mobility differences.
These variations can potentially cause conformational changes in the DNA molecules. Under
non denaturing conditions and often reduced temperature, single-stranded DNA molecules can
assume unique conformations that vary depending on their nucleotide sequences so the
conformation (structural) vary from species to species because the genetic sequences vary from
species to species and individual to individual. These conformational changes can result in
detectable differences in mobility as illustrated in Figure below. In this application note we will
review SSCP analysis, a technique that is widely used for sequence variation detection because
of its simplicity and ease of use in forensic investigation to develop the microbial community
profiling or to study the microbial profiling of crime related suspect persons and try to find out
the location of crime.
D. Amplified ribosomal DNA restriction analysis (ARDRA):
Amplified ribosomal DNA restriction analysis (ARDRA) is a simple procedure in which a
standard restriction digestion analysis is performed on PCR-amplified rDNA. This method is
also known as RFLP (restriction fragment length polymorphism). In this method, PCR
amplification of rDNA genes (16S, 23S, etc.) is first performed on a community sample.
Following this, various restriction enzymes or combinations thereof are used to digest the
amplified community DNA.The operating principle of this method is that divergences in the
rDNA gene sequences of different species will create differences in restriction sites for various
enzymes. If the correct restriction enzymes are used, what should emerge is a unique fingerprint
for each species or strain .This digested DNA is run on a gel, producing a pattern of fragment
sizes that is characteristic of the community. For single isolates or clones, the digests can be run
on regular agarose. However in studies of complex communities, the large number of DNA
fragments produced by this method can only be resolved using polyacrylamide gels.
E. Amplified fragment length polymorphism (AFLP):
AFLP analysis is a genetic mapping technique that uses selective amplification of a subset of
restriction enzyme-digested DNA fragments to generate a unique fingerprint for a particular
genome. First developed for plant studies, AFLP analysis is used for a variety of applications,
such as:
• Creation of genetic maps for new species
• Determination of relatedness among species
• Establishment of linkage groups in parentage disputed cases
• Genetic diversity and molecular phylogeny studies in microbial forensic.
The power of AFLP analysis derives from its ability to quickly generate large numbers of marker
fragments for any organism, without prior knowledge of the genomic sequence. In addition,
AFLP analysis requires only small amounts of starting template and can be used for a variety of
genomic DNA samples. The AFLP procedure consists of two amplification steps: a low-level or

October 18, 2012
preselective amplification, followed by a more selective amplification, which generates a set of
fragments that can be used as the discriminatory marker set for a particular sample.
F. Denaturing gradient gel electrophoresis:
Denaturing gradient gel electrophoresis (DGGE) is a method that separates PCR-amplified
rDNA according to differences in sequence G-C content, based on differential mobility through a
DNA-denaturing gel. In this method, PCR-amplified DNA from taxonomically differentiated
genes is run on a special polyacrylamide gel, which has embedded a gradient of DNA-denaturing
compounds, usually urea and form amide. As DNA passes through a concentration gradient (i.e.,
from low to high) of denaturant, it comes under increasing pressure to separate into single
strands. The DNA is unable to denature completely because of the presence of a GC clamp,
which is included in one of the primers for the PCR reaction. It does, however, become
increasingly denatured as it passes through the gel, which decreases its mobility. The DNA
comes to rest when it is almost fully denatured. The position along the gradient at which this
occurs is determined primarily by the relative proportions of G+C and A+T in a given amplicon,
since G-C bonds are more difficult to denature than A–T bonds. Thus, differences in sequence
between amplicons that result in differences in G-C content will cause DNA to migrate to
different positions in the gel. Properly calibrated, DGGE is sensitive enough to detect even single
base-pair differences between amplicons. DGGE is perhaps the most commonly used method of
community characterization, in a manner similar to the other PCR-based genetic fingerprinting
techniques.
G. TGGE (Temperature gradient gel electrophoresis):
There exists a variant of DGGE called temperature gradient gel electrophoresis (TGGE). TGGE
operates on the same principles as DGGE, provides approximately the same degree of
specificity, and possesses the same advantages and limitations. The only difference is that TGGE
employs a gradient based on temperature (which also denatures DNA differentially depending on
G-C content), rather than a gradient
based on chemical denaturants.
REFERENCE:
http://www.biology-questions-and-answers.com/viral-infection.html
http://www.biology-questions-and-answers.com/viruses.html
http://www.intechopen.com/books/forensic-medicine-from-old-problems-to-new-challenges/
forensic-microbiology
http://www.globalsecurity.org/wmd/intro/bio_viral.htm

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