Download I PG - VIROLOGY E-LEARNING

NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF MICROBIOLOGY
E-LEARNING
CLASS
SUBJECT
: I MSC MICROBIOLOGY
: VIROLOGY AND NANOTECHNOLOGY
SEMESTER II : PAPER VI ; VIROLOGY AND NANOTECHNOLOGY
UNIT I
History and scope of Virology. Viruses – Definitions – Structures – General properties and
Classifications. Bacterial Viruses – Introduction – taxonomy of bacterial viruses – DNA
containing Viruses – Structure – Replication and Growth kinetics of T phages (Lytic and
Lysogeny). Filamentous phages – RNA containing viruses – Structure, Replication and growth
kinetics of _ x 174 .
UNIT II
Plant viruses – RNA viruses, TMV, Cowpea, Mosaic viruses, Bromo mosaic viruses, Satellite
viruses. Double stranded DNA viruses – CaM Viruses, Single stranded DNA viruses – Gemini
virus.
UNIT III
Animal viruses – RNA viruses – Picorna virus, Herpes virus, Toga virus. RNA tumor viruses –
Retro viruses. DNA viruses – Vaccinia virus, DNA tumor virues – SV 40, Adeno viruses, emerging
viruses, Slow viruses, Prions.
UNIT IV
Techniques in virology – Cultivation of viruses – Isolation and Purification of Viruses.
Characterization and Enumeration of viruses – Quantitative assay.
UNIT V
Introduction to Nanotechnology – Scientific revolutions – Types of nanotechnology and
Nanomachines. Definition of Nanosystem- dimensionality and size dependant phenomenon;
Quantum Dots, Nanowires and Nanotubes, 2D films.
References:
Luria. S.E. Darnall. J.E. Baltimore. D. and Compare. A. 1978. General Virology, 3 ed.
Freidfelder ,D. 1995. Microbial genetics.
Grierson. D. and S.Convey, 1989. Plant Molecular Biology, 2 ed.
Hayes. W. 1968. The Genetics of Bacteria and their Viruses.
Mundahar. C.L. 1987. Introduction to plant viruses. 2 ed.
UNIT I
History and scope of Virology. Viruses – Definitions – Structures – General properties and
Classifications. Bacterial Viruses – Introduction – taxonomy of bacterial viruses – DNA
containing Viruses – Structure – Replication and Growth kinetics of T phages (Lytic and
Lysogeny). Filamentous phages – RNA containing viruses – Structure, Replication and growth
kinetics of ф x 174 .
--------------------------------------------------------------------------------------------------------------------Part A
1. Define Virus
Viruses may be defined as acellular organisms whose genomes consist of nucleic acid, and which obligately
replicate inside host cells using host metabolic machinery and ribosomes to form a pool of components which
assemble into particles called VIRIONS, which serve to protect the genome and to transfer it to other cells
2. Examples for large and small viruses
A small virus has a diameter of about 20nm - Parvovirus
A large virus have a diameter of up to 400nm – Poxviruses
3. Capsid
 The protein shell, or coat, that encloses the nucleic acid genome.
 Functions: a. Protect the viral nucleic acid. b. Participate in the viral infection. c. Share the antigenicity
4. Nucleocapsid (Nucleoprotein)
 The core of a virus particle consisting of the genome plus a complex of proteins.
 complex of proteins = Structural proteins +Non- Structural proteins (Enzymes &Nucleic acid binding
proteins)
5. Virion
 the complete infectious unit of virus particle
 Structurally mature, extracellular virus particles.
Part B
1. Write briefly on General Characteristics of Viruses
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All viruses are obligate parasites.
They multiply only within their living hosts cells and remain metabolically inert outside the host cell.
They are ultramicroscopic and can only be viewed with electron microscope (the smallest known virus is
merely 0.002 μm in diameter, while the largest ones are typically about 0.8μm in diameter).
Viruses are, actually, nucleoproteins. The proteinaceous coat (capsid) surrounds the nucleic acid, which
forms the central core of the virus particles.
The viral genetic material, the nucleic acid, may be either DNA or RNA. The two nucleic acids are never
present in a given virus.
These are the nucleic acids of the viruses which are infectious, and not the protein coat.
Viruses are usually so minute that they can easily pass through a filter, which can hold back even the
smallest bacteria.
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Viruses are easily transmitted from infected host to the healthy ones through various agencies.
Viruses are so effective that even their smallest amount can cause infection on the host successfully.
Viruses can easily be crystallized.
Viruses are considered to be host-specific and represent obligate parasitism even at genetic level.
Since the viruses have no metabolic activities of their own and utilize the metabolism of host cells,
antibiotics have no effect on them.
2. Write briefly on Structure of T-Even Phages (T4 Bacteriophage)
 DNA is packaged in the head of T4 phage.
 The tail shaft is composed of two concentric cylinders. The outer cylinder is called tail
sheath, and it will contract upon infection.
 The hexagonal baseplate is made of 22 kinds of proteins.
 The tail fibers are responsible for recognizing the receptor in the outer membrane of
the host cell.
 Virions not enveloped; tailed; head.
 Head separated from tail by a neck,
 tail complex, consisting of a central tube and a contractile sheath, provided with a
collar, base plate, 6 short spikes and 6 long fibers.
 Nucleocapsids isometric to quasi-isometric elongated; 65-115 nm in diameter; 95-111 nm
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long; 65-80 nm in diameter.
Symmetry icosahedral. Nucleocapsids appear to be angular.
152 capsomers per nucleocapsid. Tail contractile; 80-113 nm long; 16 nm wide.
 The T-even phage is characterized by the presence of a hexagonal head about
900 Å wide. It consists of dsDNA molecule protected by a protein coat made up of
numerous facets. The DNA molecule, measuring about 52,000 Å in length, is
coiled and packed inside the head.
1. Head
2. Protein Sheath
3. Coiled DNA
4. Collar
5. Central Core 6. Protein Sheath (Helical)
7. Tail
8. End Plate
9. Tail Fibres
10. Hexagonal Plate
11. Spike
Part C
1. Explain detail on Life-Cycle (Multiplication or Infection Cycle) of T-Even Phages
Virulent phages do not integrate their genetic material into the host cell chromosome and usually kill the
host cells (lytic infection) (e.g. T-phages of E.coli).
Temperate phages may integrate into the host DNA, causing LYSOGENY.
The infection cycle of T-even bacteriophage lasts about 20 minutes, culminating in lysis (bursting-open) of the host
cell, E. coli. The whole process can be classified into, (i) adsorption or .infection. (ii) penetration or injection, (iii)
the eclipse or the latent period (iv) maturation and (v) lysis or release.
(i) Adsorption or infection
Attachment of the virus particle onto the surface of the host cell is
adsorption or infection. The virus particles possess one or more proteins on
the outside that interact with cell surface components called receptors; the
receptors are normal surface components of the host (e.g., proteins,
carbohydrates, glycoproteins, lipids, lipoproteins, etc.). Infact, these are the receptors that determine which cells
will be susceptible to infection. In the absence of the receptor site, the virus can not adsorb and hence can not
infect. If the receptor site is altered, the host may become resistant to virus infection.
(ii) Penetration or Injection
This process is very interesting and has been studied and beautifuIly elucidated by B.Kellenberger. After the tail
fibres get adsorbed, an enzyme-system is supposed to make a pore or hole in the cell wall of the host. It is believed
that the enzyme-system consists of a phage-lysozyme, which is synthesized during the multiplication of the parent
phage inside the host cell and its molecules remain attached to the extreme tip of the tail-fibres of the new
progeny phages.
This enzymes system becomes active when the released phage particles infects the new host cell.
However, the tail-fibres attached on the surface of the host cell bend to bring the end-plate in contact with the cell
wall surface. Now, the protein sheath of the tail longitudinally contracts pushing the central tubular core through
the pore inside the wall of the host cell and the phage DNA molecule is released or injected into the cytoplasm.
After the DNA is released, the empty protein coat becomes of no use.
(iii) The Eclipse or the Latent Period
When the DNA molecule is released in the host cytoplasm, it is not degraded by the nuclease enzymes of the host
cell. It has been studied, particularly in T4, phage, that the phage DNA contains glucosylated hydroxymethyl
cytosine instead of cytosine, which prevents the nucleases of the bacterium from degrading the phage DNA. The
phage DNA, first makes the host cell immune against infection by genetically similar phage particles. Secondly, it
immediately takes over the charge of the cell machinery and suppresses all cellular activities such as synthesis of
cellular DNA, RNA, proteins, etc. This is the parasitism of a virus at the genetic level.
This suppression is short lived and the cell machinery of protein synthesis starts functioning under the
control of viral DNA in the place of
cellular-DNA.
New
messenger-RNA
molecules are synthesized very rapidly
and a series of new enzymes, namely,
‘early proteins’ is synthesized viral DNA
molecules direct the formation of new
type of proteins, namely, ‘late proteins’.
Majority of the late proteins are viral coat
proteins, whereas some are phage
lysozyme. The viral coat proteins
constitute the sheath of the phage and
the phage-lysozyme later help in the
injection process.
(iv) Maturation
Assembly of the various components to
constitute a new phage particle within
the host cell is called maturation. Head the tail formation start separately, the protein components aggregate
around the DNA and form the Head of the phage. End-plate is formed first followed by the formation of tubular
core. Tail fibres are formed later. Hundreds (about 200) of new phage particles are produced from each bacterium
by the time of lysis.
(v) Lysis or Release
After the production of new bacteriophages, the host bacterial cell bursts open and the phage particles are
released. Bursting open of the host bacterial cell is called 'lysis'.
Life-cycle (Multiplication) of Lambda Phage – LYSOGENY CYCLE
The bacteriophage is first adsorbed on the host wall surface and its DNA is injected into the bacterial cell
cytoplasm. The viral DNA, instead of staring lytic cycle, gets inserted into the bacterial DNA and travels through
many generations by means of the successive division of the cell. Under certain conditions, the inserted viral DNA
may get dissociated form the bacterial DNA, and start functioning as virulent phage culminating in the lysis of the
host cell. Such conversion of temperature phage (especially prophage) into the virulent phage is referred to as
‘induction’, which can be artificially achieved by treatment of the bacterial cells with ultraviolet radiation or with
hydrogen peroxide.
UNIT II
Plant viruses – RNA viruses, TMV, Cowpea, Mosaic viruses, Bromo mosaic viruses, Satellite
viruses. Double stranded DNA viruses – CaM Viruses, Single stranded DNA viruses – Gemini
virus.
--------------------------------------------------------------------------------------------------------------------Part A
1. TMV
Tobacco mosaic virus (TMV) belongs to Tobamovirus group of plant viruses. It is a historically important and
thoroughly studied virus, which causes mosaic disease on tobacco plants and others like tomato, etc. W.M. Stanley
(1935) for the first time isolated this virus in its crystalline form and he was awarded Nobel Prize for this work.
2. Treatment of TMV
One of the common control methods for TMV is sanitation, which includes removing infected plants, and washing
hands in between each plant treatment. Crop rotation should also be employed to avoid infected soil/seed beds
for at least 2 years. As for any plant disease, looking for resistant strains against TMV may also be advised.
Furthermore, the cross protection method can be administered, where the stronger strain of TMV infection is
inhibited by infecting the host plant with mild strain of TMV, similar to the effect of a vaccine.
3. Gemeni virus
The most surprising features of this virus group are the small capsid size, 18-20 nm x 30 nm, their geminate (paired
particles) morphology, which sets them apart from all other classes of viruses, and the unexpected covalently
closed circular topography of the single stranded DNA which is in the molecular weight range of 7 x 105 to 9 X 105.
4. BMV
Brome mosaic virus (BMV) is a small (27 nm, 86S), positive-stranded, icosahedral RNA plant virus belonging to the
family Bromoviridae of the alphavirus-like superfamily.
Part B
1. Briefly explain Structure of Tobacco Mosaic Virus (TMV)
TMV is a simple rod-shaped helical virus consisting of centrally located singlestranded RNA (5.6%) enveloped by a protein coat (94.4%). The rod is considered to
be 3,000 Å in length and about 180 Å in diameter. The protein coat is technically
called 'capsid'. There are 2,130 sub-units, namely, capsomeres in a complete helical
rod and 49 capsomeres on every three turns of the helix; thus there would be about
130 turns per rod of TMV. The diameter of RNA helix is about 80 Å and the RNA
molecule lies about 50 Å inward from the outer-most surface of the rod. The central
core of the rod is about 40 Å in diameter. Each capsomere is a grape like structure
containing about 158 amino acids and having a molecular weight of 17,000 dalton.
The ssRNA is little more in length (about 3300 Å) slightly protruding from one end of
the rod. The RNA molecule consists of about 7300 nucleotides; the molecular
weight of the RNA molecule being about 25,000 dalton.
2. Briefly explain Structural Features of Geminiviruses
The most surprising features of this virus group are the small capsid size, 18 -20 nm x 30 nm, their geminate (paired
particles) morphology, which sets them apart from all other classes of viruses, and the unexpected covalently
closed circular topography of the single stranded DNA which is in the molecular weight range of 7 x 10 5 to 9 X 105.
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All geminiviruses recognized so far have a single major coat protein subunit in the range of 2.7-3.4 x 104
daltons. The genome of the geminiviruses consists of either one or two circular, single stranded DNA
molecules.
The single stranded viral DNA, 2.6 to 3.0 kb long, is converted in the nucleus of plant cells into a double
stranded replicative form by an as yet unknown mechanism.
Many copies of the replicative form of a geminivirus genome accumulate inside the nuclei of infected cells.
There is 110 evidence to date for a reverse transcription step in geminivirus replication.
Bean golden mosaic virus (BGMV) DNA was found to be 2510 nucleotides long; if this was the complete
genome, it is less than half the length of any other known autonomously replicating plant virus.
By comparing the single stranded DNA of the virus particle with the viral double stranded DNA found in
infected plants, it was found that the nucleotide sequence had a complexity twice that expected given the
physical size of the viral DNA.
This indicates that the BGMV DNA is heterogeneous and has a divided genome consisting of two DNA
molecules of approximately the same size, but differing in genetic content.
It would appear that geminiviruses consist of two populations of paired particles, differing only in the
nucleotide sequence of the DNA molecules they contain. Transmission of the virus in nature occurs by
leafhoppers or the tropical whitefly.
3. Write a detail account on Structure and Properties of CaMV
The GaMV particles are spherical, isometric, or icosahedral, about 50 mm in diameter, contain circular double
stranded DNA of about 8 kb, and can be isolated from an inclusion body using urea and nonionic detergents. There
are probably four struc1ural polypeptides but only two account for over 90% of the viral protein.
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The major components have mol. wts of 37,000 and 64,000 and are present in a molar ratio of 5 to 1. From
the proportion of these polypeptide species and the mol. wt of the virus particle, the most likely structure is
an outer shell of 420 molecules of the 37,000 species surrounding a core of 60 molecules of the 64,000
species.
The two remaining polypeptides have much higher mol. wts (96,000 and 88,000) and may be glycoproteins.
Their function is not known. The DNA molecule is about 8 kbp long and several varieties (totaling 50,000
bases) have been sequenced.
The DNA exists in linear, open, circular and twisted or knotted forms; however, none of the circular forms is
covalently closed due to the presence of site specific single strand breaks.
These nuclease sensitive single strand breaks are not true gaps but short oligonucleotide overlapping regions,
having sequence complementarity, and thus forming short triple stranded structures with a fixed 5'-end.
There are three such sites, one (1) in the minus (coding or transcribed) strand yielding the large fragment and
overlapped by eight residues.
The other two (2 and 3) are in the plus (noncoding or nontranscribed) strand yielding the and t fragments
having 18 and 15 residue overlaps respectively. One of the plus strand discontinuities is dispensable and.
none is required for infection, as virus DNA previously cloned in bacteria and. lacking the "gaps" is as
infectious as native DNA.
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The whole base sequence has been determined and from the sequence one can identify eight open reading
frames (ORFs). So far genetic evidence suggests that ORF VI codes for the matrix protein of the inclusion
body. ORF IV codes for a coat protein. ORFs II and VII seem not to be necessary for a successful infection.
Sequence analyses of the DNA at these discontinuities in GaMV have shown that they in fact consist of short
overlaps of 6-18 nucleotides so that there is a terminal redundancy in one strand of the DNA at the site of the
discontinuity.
Presumably as a result of these overlaps, the DNA can be isolated in a variety of twisted circular forms
resembling supercoils and also as a linear form due to breakage. The function of the discontinuities in the
DNA is not clear, although they are not required for infectivity.
Sequence data obtained from GaMV has revealed six major and two minor tightly packed, potential coding
regions distributed between three reading frames.
Transcription of CaMV is found to be asymmetric, with only the strand producing stable transcripts. The
mechanism of replication seems to be as follows: The infecting GaMV DNA enters the plant nucleus, where
the single stranded overlaps are digested and the gaps ligated to give a supercoiled minichromosome.
The function of this minichr0mosome is to act as a template for plant nuclear RNA polymerase II. The
transcript thus formed is transported to the cytoplasm where it is either translated or replicated by reverse
transcription. A site 600 b downstream of the promoter of the large transcript binds the proposed primer of
reverse transcription, methionine tRNA.
The RNA transcript is then copied into the minus strand DNA. Synthesis of the plus strand DNA starts at two
primer binding sites near "gaps" 2 and 3. From gap 2 synthesis proceeds to the 5'-end of the minus strand
DNA, whereas synthesis from gap 3 continues to gap 2. This DNA molecule gets packed into virus particles or
reel1ters the nucleus and undergoes another round of transcription and/or translation/replication.
Two major RNA transcripts are found inside infected cells. One covers the sequence of ORF VI. Another and
larger one contains the base sequence of the whole genome. (The RNA polymerase goes all the way around
the template strand and then continues for a short distance, repeating the first transcribed segment.) It is
likely that the larger RNA acts as a polygenic messenger RNA.
Part C
1. Write a detail account on Purification of Plant viruses
Purification or, as it is usually called, isolation of viruses is necessary to know about their structure and other
properties. By employing the method of purification, a virus is finally obtained in its pure form as a colourless
pellet in a test tube and may be used for various purposes. Following are the steps involved in virus purification
(isolation)
1. Infected leaves are thoroughly homogenized in water or preferably in phosphate, borate or citrate buffer in
an electric grinder or in a mortar with pestle.
2. Tissue homogenate is strained through a peice of muslin cloth (or cheese cloth). Crude sap which comes out
and contains virus is collected and then poured into centrifuge tube. The tube is spun at low-speed (3000-17000
g). As a result, the crude sap differentiates into supernatant and a pellet. The pellet is discarded and the
supernatant with virus is collected.
3. The supernatant with virus is poured into centrifuge tube. The tube is placed in fixed-angle-rotor of
ultracentrifuge and spun at high speed (40000-150000 g). After the tube settles, the virus sediments and forms
tiny pellet at the bottom of the tube and a supernatant over it. Supernatant is discarded and the pellet of virus is
mixed with a buffer and stirred with rod so that it resuspends in buffer.
1.
Low and high speed centrifugation steps are repeated 2-3 times and the virus is purified by density
gradient centrifugation, the most frequently used technique. A tier of layer of sucrose solutions of
different concentrations (e.g., 10-40%), and hence densities, is formed in the centrifuge tube; layer at
the bottom being the most dense and one at the top the least dense with layer of intermediate
concentrations. Virus suspension is placed at top of the top-most layer and centrifuge tube centrifuged
in swimming-bucket rotors at high speed ultracentrifuge.
2.
When settled, virus particles move together as a band in gradient solution of sucrose. The virus-band is
collected as separate fraction through puncture at the bottom of the centrifuge tube. The virus-fraction
is placed in cellulose dialysis tubing and sucrose is removed by dialysis in buffer solution or water. Thus,
the virus is obtained in pure form.
The concept of purity of viruses is an optional one because the virus preparation obtained after
purification is, however, rarely absolutely pure as it usually contains some impurities. For practical
purposes a virus preparation is considered to be pure if its properties (e.g.. amino acid composition,
nucleotide composition, percentage of protein, sedimentation profile etc.) do not change upon further
purification. However, the purification of a virus is always done with some particular experimental work in
mind so that the degree of purity is tested with reference to that work.
2. Write briefly on BMV
Brome mosaic virus (BMV) is a small (27 nm, 86S), positive-stranded, icosahedral RNA plant virus belonging to the
family Bromoviridae of the alphavirus-like superfamily.
BMV commonly infects Bromus inermis (see Bromus) and other grasses, can be found almost anywhere
wheat is grown, and thrives in areas with heavy foot or machinery traffic. It is also one of the few grass viruses that
infects dicotyledonous plants; however, it primarily infects monocotyledonous plants, such as barley and others in
the family Gramineae.
BMV was first isolated in 1942 from bromegrass (Bromus inermis) (Lane, 1974), had its genomic
organization determined by the 1970s, and was completely sequenced with commercially available clones by the
1980s (Ahlquist et al., 1981; Lane, 2003).
The alphavirus-like superfamily includes more than 250 plant and animal viruses including Tobacco mosaic
virus, Semliki forest virus, Hepatitis E virus, Sindbis virus, and arboviruses (which cause certain types of
encephalitis) (Sullivan & Ahlquist, 1997; Lampio, 1999). Many of the positive-strand RNA viruses that belong to the
alphavirus family share a high degree of similarity in proteins involved in genomic replication and synthesis
(Ahlquist et al., 1985; French & Ahlquist, 1988). The sequence similarities of RNA replication genes and strategies
for BMV have been shown to extend to a wide range of plant and animal viruses beyond the alphaviruses,
including many other positive-strand RNA viruses from other families (Ahlquist, 1992). Understanding how these
viruses replicate and targeting key points in their life cycle can help advance antiviral treatments worldwide.
BMV consists of a genome that is divided into three 5' capped RNAs. RNA1 (3.2 kb) encodes a protein
called 1a (109 kDa), which contains both an N-proximal methyltransferase domain and a C-proximal helicase-like
domain. The methyltransferase domain shows sequence similarity to other alphavirus m7G methyltransferases
and guanyltransferases, called nsP1 proteins, involved in RNA capping (Ahola & Ahlquist, 1999). RNA2 (2.9 kb)
encodes the 2a protein (94 kDa), the RNA-dependent RNA polymerase, responsible for replication of the viral
genome (Ahlquist, 1992; Sullivan & Ahlquist, 1997). The dicistronic RNA3 (2.1 kb) encodes for two proteins, the 3a
protein (involved in cell-to-cell migration during infection) and the coat protein (for RNA encapsidation and
vascular spread), which is expressed from a subgenomic replication intermediate mRNA, called RNA4 (0.9 kb). 3a
and coat protein are essential for systemic infection in plants but not RNA replication (Sacher & Ahlquist, 1989;
Sullivan & Ahlquist, 1997; Diez et al., 2000).
UNIT III
Animal viruses – RNA viruses – Picorna virus, Herpes virus, Toga virus. RNA tumor viruses –
Retro viruses. DNA viruses – Vaccinia virus, DNA tumor virues – SV 40, Adeno viruses, emerging
viruses, Slow viruses, Prions.
--------------------------------------------------------------------------------------------------------------------Part A
1. Herpes simplex viruses 1 and 2
Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) initially infect epithelial cells of the oral or genital
mucosa, the skin or the cornea. The virus may enter neurones and may be transported to their nuclei, where they
may establish latent infections. HSV-1 commonly infects via the lips or the nose between the ages of 6 and 18
months. A latent infection may be reactivated if, for example, the host becomes stressed or immunosuppressed.
Reactivation results in the production of virions, which in about 20–40 per cent of cases are transported within the
neurone to the initial site of infection, where they cause productive infection in epithelial cells, resulting in a cold
sore. Occasionally there may be serious complications such as encephalitis, especially in immunocompromised
hosts. HSV-2 is the usual causative agent of genital herpes, which is a sexually transmitted disease. In newborn
babies infection can result in serious disease, with a mortality rate of about 54 per cent. Although the face and the
genitals are the normal sites of infection for HSV-1 and HSV-2, respectively there are increasing numbers of cases
where HSV- 1 infects the genitals and HSV-2 infects the face.
2. Epstein-Barr virus
Epstein-Barr virus (EBV) is transmitted in saliva. Epithelial cells are infected first then the infection spreads
to B cells, which are the main host cell type for this virus. More than 90 per cent of people become infected with
EBV, usually during the first years of life, when infection results in few or no symptoms. In developed countries
some individuals do not become infected until adolescence or adulthood. A proportion of these individuals
develop infectious mononucleosis (glandular fever), commonly called ‘the kissing disease’ by doctors. EBV is
associated with a number of tumours in man
3. Variola and Vaccina
Variola - Causative agent of small pox
Vaccina – Laboratory altered pox virus, used for vaccination against small pox
Part B
1. Write briefly on herpesvirus virion
Herpesviruses have relatively complex virions composed of a large number
of protein species organized into three distinct structures: capsid, tegument
and envelope. The virus genome is a linear dsDNA molecule, which varies in
size within the herpesvirus family from 125 to 240 kbp. The DNA is housed in
the capsid, which is icosahedral, and the capsid is surrounded by the
tegument. The HSV-1 tegument contains at least 15 protein species and
some virus mRNA molecules. The envelope contains a large number of
spikes (600–750 in HSV-1) composed of ten or more glycoprotein species.
There are several different sizes of spike. A number of schemes have
evolved for the nomenclature of herpesvirus proteins, with the result that
an individual protein may be referred to in the literature by two or more different names. Most of the structural
proteins are commonly named VP (virus protein). In HSV-1 the most abundant proteins in the capsid and the
tegument are VP5 and VP16, respectively. In the envelope there are at least 12 species of glycoprotein, each of
which is prefixed ‘g’, for example gB, gC and gD. The capsid is constructed from 162 capsomeres, 12 of which are
pentons and the remainder of which are hexons
2. Write briefly on emerging virus
A virus is an infectious agent that is incapable to grow or reproduce outside a host cell. It itself can cause severe
problems with health and even lead to death in a long term or short term period whereby it reproduces itself
inside a human body.
With a slight knowledge on the effects of what virus area capable of, we would like to talk about new viruses or
evolution of the viruses, in other words, Emerging virus.
The factor that contribute to emerging viruses are usually mutation of the virus which we would term it
Spontaneous evolution of virus.
 Many viruses, in particular RNA viruses, have short generation times and relatively high mutation rates.
 This elevated mutation rate, when combined with natural selection, allows viruses to quickly adapt to
changes in their host environment.
For example, a series of outbreaks of virus within the 6 years are as follows;
Year 1999 ( West Nile Virus)
West Nile virus is an emerging virus that first appeared in North America during the summer of 1999 in New York
City. There were seven deaths associated with this event. Surveillance reports indicate that the virus had been
spreading south and west and in 2002, had been reported in 42 states and the District of Columbia. As of
September 2002, there were 2121 total human cases reported, inducing 104 deaths. The fatality rate for the West
Nile virus is very low and the majority of individuals will have no clinical symptoms; however, individuals at most
risk for more serious form of the disease are the elderly, the immunocompromised, and young individuals. The
virus is spread by certain mosquito species and certain populations of birds serve as the reservoir hosts. Because
person-to-person transmission does not occur, humans are therefore considered dead-end hosts. Confirmation of
cases West Nile virus infections in humans are determined based on clinical and laboratory findings.
September 1998 – April 1999 (Nipah Virus Outbreak)
Nipah virus is a newly recognized zoonotic virus. The virus was 'discovered' in 1999. It has caused disease in
animals and in humans, through contact with infectious animals. The virus is named after the location where it was
first detected in Malaysia. Nipah is closely related to another newly recognized zoonotic virus (1994), called
Hendra virus, named after the town where it first appeared in Australia. Both Nipah and Hendra are members of
the virus family Paramyxoviridae. Although members of this group of viruses have only caused a few focal
outbreaks, the biologic property of these viruses to infect a wide range of hosts and to produce a disease causing
significant mortality in humans has made this emerging viral infection a public health concern.
November 2002 – July 2003 (SARS Outbreak)
Severe acute respiratory syndrome (SARS) is a respiratory disease in humans which is caused by the SARS corona
virus. There has been one near pandemic to date, between November 2002 and July 2003, with 8,096 known
infected cases and 774 deaths worldwide being listed in the World Health Organization's (WHO) 21 April 2004
concluding report. Within a matter of weeks in early 2003, SARS spread from the Guangdong province of China to
rapidly infect individuals in some 37 countries around the world. Mortality by age group as of 8 May 2003 is below
1 percent for people aged 24 or younger, 6 percent for those 25 to 44, 15 percent in those 45 to 64 and more than
50 percent for those over 65. For comparison, the case fatality rate for influenza is usually around 0.6 percent but
can rise as high as 33 percent in locally severe epidemics of new strains. The mortality rate of the primary viral
pneumonia form is about 70 percent.
Year 2008 (Avian Influenza virus)
Avian influenza, also known as bird flu, is caused by avian influenza virus, which belongs to the family
Orthomyxoviridae, and affects most domestic and wild bird species. The influenza viruses are divided on the axiom
of their antigenic nature into types A, B and C. Only type A influenza viruses are of diversion to veterinarians,
whereas types B and C are usually found in humans. Avian influenza viruses are further sorted by their surface
proteins, haemagglutinin and neuraminidase. There are 15 different haemagglutinin (H1-15) and nine
neuraminidase (N1-9) subtypes based on serological testing with the haemagglutinin-inhibition and neuraminidase
inhibition tests, respectively. The virulence of the avian influenza viruses varies broadly from very low to very high
and signs of disease range from mild respiratory to viscera-tropic, multi-systemic infections with catastrophic
consequences. Low pathogencity avian influenza (LPAI) viruses can be any of the 15 haemagglutinin and nine
neuraminidase subtypes, whereas highly pathogenic avian influenza (HPAI) viruses have only been of the H5 and
H7 haemagglutinin subtypes. Generally, antibodies against one of the haemagglutinins offer little or no protection
against a virus with a different haemagglutinin subtype.
The virus remains viable for long periods in tissues and faeces. Various physical and chemical treatments can
inactivate the virus, such as temperatures of 56°C for 3 hours or 60°C for 30 min, acidic pH, oxidizing agents,
sodium dodecyl sulphate, lipid solvents, ß-propiolactone, as well as formalin and iodine disinfectants.
Part C
1. Write a detail account on Morphology, Pathogenesis and Lab diagnosis of Adeno virus
Adenovirus, a DNA virus, was first isolated in the 1950s in adenoid tissue–derived cell cultures, hence the name.
These primary cell cultures were often noted to spontaneously degenerate over time, and adenoviruses are now
known to be a common cause of asymptomatic respiratory tract infection that produces in vitro cytolysis in these
tissues.
Morphology of the Adenovirus
Adenoviruses have a characteristic morphology: they are medium-sized
(90–100 nm), nonenveloped icosohedral viruses (Stewart et al., 1993) that
contain double-stranded DNA. The genome is 36–38 kb. The viral genome is a
linear, double-stranded DNA, 36–38 kb long, with the dCMP covalently linked at
the 5' end of the adenoviral DNA to a 55 kD terminal protein (TP) (Rekosh et al.,
1977). The viral genomic DNA is flanked by 100–140 bp inverted terminal repeats
(ITRs), which serve as replication origins.
The capsid exhibits isometric icosahedral symmetry. Its surface structure
has a regular pattern and the capsomere arrangement is clearly visible. Surface
projections are distinct, including one or two filaments protruding from the 12
vertices, as well as a central cavity. The viral capsid is made of 252 protein
components and its outline is hexagonal with three major types of protein
components – penton bases, fibers and hexons. There are 240 hexons and 12
pentons on each adenoviral particle.
Penton base proteins are toxic factors that affect cultured cells by making them detach. Fiber proteins are
hemagglutinins and major neutralizing epitopes that are subtype specific. Hexon proteins are group-specific
antigenic determinants and type-specific neutralizing epitope. Penton bases are tightly associated with one or two
fibers; fibers interact to form a shaft of 9-77.5 nm with a distal knob. The core of each fiber consists of a
nucleoprotein complex.
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Adenoviruses were first isolated in 1935 from human adenoid tissues.
Since then, at least 49 distinct antigenic types have been isolated from humans and many other types from
animals.
All human serotypes are included in a single genus within the family Adenoviridae.
Adenoviruses are divided into six groups (A to F) based on
– physical,
–
chemical
–
biological properties
Antigenic structure divides adenoviruses into:
- 49 serotypes:
- About 1/3 of the 49 known human serotypes are responsible for most cases of Adenovirus disease.
Adenoviruses spread by:
– direct contact
– respiratory droplets
– feco-oral route.
Pathogenesis
The recognized diseases of adenoviruses predominantly involve the respiratory tract, the GI tract and the eye.
Virus may be introduced through contact, respiratory droplets or ingestion. The association of particular types with
specific disease syndromes is striking. After recovery of illness, adenoviruses may maintain latent persistent
infections in the tonsils, the adenoids, and other lymphoid tissues of man, and they are readily activated. (Most
persons are infected with one or more types of adenovirus before the age of 15, 50 to 80% of tonsils removed
surgically yield an adenovirus when cultured in vitro). Ad1, Ad2 and Ad5, members of subgenus C, persist in tonsils
for several years. Shedding of infectious virus in the stools for at least 2 years have been documented. Adenovirus
strains can also be secreted in the urine.
Clinical Syndrome
1. Respiratory Disease in Children - Adenoviruses are responsible for 5% of acute respiratory infections in
children under the age of 4 years, whereas they account for 10% of hospitalized respiratory infections in
this age group. Adenoviruses can also cause laryngotracheobronchitis, but the pneumonias that occur in
young children are the most serious manifestations. These may occur as a consequence of infection with
the endemic Ad2 and Ad5, among which certain strains can be more aggressive than others. In particular,
Ad 3 and Ad7 may also create severe problems. Adenoviruses have been reported to account for 10% of
pneumonias of childhood. The severity of symptoms is related to overcrowding. In the winter of 1959,
3398 cases of adenovirus pneumonia with a fatality rate of 15.5% were seen at the Peking children's
hospital. Among the survivors of severe respiratory infections, residual lung damage due to secondary
obliterative bronchiolitis has been reported. Bronchiectasis and abnormal lung function tests have also
been reported as sequelae.
2.
Pharyngoconjunctival Fever - This disease is characterized by conjunctivitis, fever, pharyngitis and
adenoidal enlargements. This is frequently associated with swimming pools. Adequate levels of chlorine
are usually sufficient to inhibit outbreaks.
3.
Acute Respiratory Disease (ARD) in Military Recruits - ARD is usually caused by Ad4, Ad7, and Ad21,
although Ad14 has been reported from Holland. In general, outbreaks do not involve seasoned troops but
cause a high morbidity among newly enlisted troops. Adenovirus infections among healthy civilian adults
are less common. The crowding of people, allowing repeated exposure to highly infectious doses, and the
strenuous physical exercise may account for the unusually high degree of severe infections. ARD usually
appears during the third week in training. Characteristic symptoms include fever, malaise, sore throat,
hoarseness and cough. Pneumonia develops in around 10% of cases.
4.
Pertussis - Adenoviruses can be isolated frequently (39%) of patients infected with B. Pertussis. The
contribution, if any, of adenoviruses to the pertussis syndrome is not known.
5.
Infections of the Eye - Acute follicular conjunctivitis, which is part of the syndrome of
pharyngoconjunctival fever, can also occur as a separate entity. The disease is also associated with
swimming pools. Epidemic keratoconjunctivitis is a distinctly different syndrome. This syndrome is
characterized by an aggressive conjunctivitis, pain, photophobia and lymphadenopathy followed by the
development of superficial punctate keratitis, corneal opacities may last for several years. In 1941, more
than 10,000 cases occurred in the marine shipyards of Pearl Harbour ("Shipyard eye"). Adenovirus
keratoconjunctivitis mainly affects males in Western countries or children in Asia and may last for 4 to 6
weeks. Antibody to Ad 8 is uncommon in American school children, whereas approximately half of the
Japanese or Taiwanese children have such antibodies.
6.
Acute Haemorrhagic Cystitis - This syndrome occurs predominantly in 6 to 15 year old boys. The
syndrome consists of acute dysuria with haematuria and is mainly associated with Adenovirus 11. Its
significance lies in the potential of being confused with more serious diseases of the kidney such as
glomerulonephritis.
7.
Infections of the gut - Adenoviruses are associated with 4-15% of all children hospitalized with viral
gastroenteritis. Gastroenteritis may be a sign of a systemic infection such as those caused by Ad3 or Ad7.
They may cause both respiratory and diarrhoea in a child with high fever. However, the enteric
adenoviruses Ad40 and Ad41 are associated with 2/3rds of cases of adenovirus-associated diarrhoea.
Several reports have appeared implicating adenoviruses in mesenteric adenitis and intussusception of the
gut.
8.
Meningitis - Adenoviruses may be infrequent cause of meningitis. Ad3 and Ad7 account for two-thirds of
all adenovirus-associated meningitis.
9.
Adenovirus infection in immunocompromised patients
b. Severe combined immunodeficiency (SCID) and immunocompromised hosts. - Children with SCID
are prone to develop severe infections with the most frequently occurring persistent
adenoviruses. Pneumonia and hepatitis, with a high mortality is seen.
c. AIDS - latent infections of the kidneys are occasionally seen in patients with AIDS and can be
readily isolated from the urine. Members of subgenus D are often seen in the stools of AIDS
patients.
d. Bone marrow transplant recipients - These patients are vulnerable to activation of all latent DNA
viruses. Adenovirus infections have been demonstrated in 8% of bone marrow transplant
recipients. There is evidence that adenovirus infections are responsible for substantial mortality
but more work needs to be done in order to assess the precise role of adenoviruses in bone
marrow transplant patients.
Laboratory Diagnosis
 Direct detection:
 Isolation
 Serology
Direct detection
 Virus particle by EM can be detected by direct examination of fecal extracts
 Detection of adenoviral antigens by ELISA.
Enteric Adenoviruses
 Detection of adenoviral NA by Polymerase chain reaction: can be used for diagnosis of Adenovirus
infections in tissue samples or body fluids.
Isolation
 Isolation depending on the clinical disease, the virus may be recovered from throat, or conjunctival swabs
or and urine.
 Isolation is much more difficult from the stool or rectal swabs
Serology
 Haemagglutination inhibition
 Neutralization tests can be used to detect specific antibodies following Adenovirus infection.
Treatment and Prevention
There is no specific antiviral chemotherapy against adenoviruses at present. Idoxuridine and ARA-A had been
tried in the treatment of keratoconjunctivitis but were unsuccessful. Antivirals have not been tried for the
adenovirus-induced respiratory syndromes. When these diseases occur, they are difficult to distinguish from
similar diseases caused by a variety of RNA viruses. Swimming pool-associated conjunctivitis can be prevented
with adequate levels of chlorine in the water.
 Careful hand washing is the easiest way to prevent infection.
 Disinfection of Environmental surfaces with hypochlorites.
 The risk of water borne outbreaks of conjunctivitis can be minimized by chlorination of swimming pools.
 Epidemic keratoconjunctivitis can be controlled by strict asepsis during eye examination.
UNIT IV
Techniques in virology – Cultivation of viruses – Isolation and Purification of Viruses.
Characterization and Enumeration of viruses – Quantitative assay.
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Cytopathic effect (CPE)
The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the
host cell due to infection are known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell rounding,
disorientation, swelling or shrinking, death, detachment from the surface, etc.
Part C
1. Write a detail account on Cultivation of viruses
(i) In Animal Cells
Suitable living mammals (such as sheep or calves or rabbits) are selected for cultivation of viruses. The
selected animals should be healthy and free from any communicable diseases. The specific virus is
introduced into the healthy animals. The site of administration varies according to the type of virus is
allowed to grow in the living animal. At the end of incubation period, the animals are slaughtered and
washed
thoroughly
and
viruses
are
obtained
from
them.
(ii) In Chick-Embryo
The animal viruses can be successfully cultivated using chickembryo technique. In this method fertile hen eggs are selected.
Eggs must not be more then 12 days old. To prepared the egg
for virus cultivation, the shell surface is first disinfected with
iodine and penetrated with a small sterile drill. After inoculation,
the drill hole is sealed with gelatin and the egg is then incubated.
Viruses may be able to region. For convenience, the mayxoma
virus grows well on the chorioallantoic membrane, whereas the
mumps virus prefers the allantoic cavity., The infection may
produce a local tissue lesion known as pock, whose appearance
often is characteristic of the virus.
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Largely replaced by use of TC
Still useful in cultivation and detection of some viruses, flu
Three parts of the egg are of use
o A The amniotic cavity:
 Surrounds the embryo & lined by a single layer of epithelial cells.
 The amniotic fluid bathes the external surface of the embryo and comes into contact
with the respiratory and alimentary tracts.
o B The allantoic cavity:
 Comprises an outgrowth of the hind-gut of the embryo and is lined with endoderm.
Both are useful for the cultivation of viruses, particularly
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orthomyxoviruses (e.g., influenza) and paramyxoviruses (e.g., mumps).
Membranes major source of cells in which virus growth occurs, but the embryo may also
become infected.
The allantoic cavity is routinely used because it is technically much simpler to inoculate.
However, some viruses (e.g., human influenza isolates) may need to be egg-adapted by growth in the
amniotic cavity before they will grow efficiently in the allantoic cavity; the reason for this is not known.
C The chorio-allantoic membrane : The membrane consists of an outer layer of stratified epithelium
which constitutes the respiratory surface of the egg, and an inner layer of endoderm (the lining of the
allantoic cavity). The membrane may be used as a cell sheet provided it is first dropped away from the
shell membrane. Dermatropic viruses (poxviruses and some herpes viruses) will grow on this membrane,
and at low concentrations, will give discrete foci of infection which consist of centres of cell proliferation
and necrosis (pocks). The membrane may therefore be used to assay these viruses. In addition, different
viruses cause pocks of different colour and morphology, and this is of diagnostic value for distinguishing
between different poxviruses.
(iii) In Vitro Culture (Tissue Culture Technique)
More recently developed in vitro cultivation of animal viruses has eliminated the need to kill the animals.
This technique has become possible by the development of growth media for animal cells and by the
availability of antibiotics which prevent bacterial and fungal contaminations in cultures. Cultivating animal
viruses using tissue culture technique involves following three main step:
Monolayer Preparation- Live tissues of vital organs (e.g., heart or kidney) are taken and the cells are
separated from the tissue by digesting the intracellular cement substance with dispersing agents such as
trypsins or collagenase or ethylenediaminetetraacetic acid (EDTA). The cell suspension is passed through
screen filters so that the coarse particles are removed from the separated cells. The cells are washed free of
dispersing agents. The cells are centrifuged if required and resuspended in nutrient medium contained in
glass or plastic vessels. The composition of medium and other conditions of incubation depends on the type
of cells used. Upon incubation the cells quickly settle and attach firmly to the bottom of the flask. If
undisturbed, these cells grow and spread to form monolayers.
Clonal Cell Line Preparation - The monolayer cells are first removed and washed with saline solution
devoid of calcium and magnesium ions and then added to the dilute solution of EDTA (1 : 3000) to chelate
intracellular magnesium or calcium ions. After sometime, the loosened cells are shaken and resuspended in
growth medium in fresh culture vessels and incubated. The cells are cultivated under 5% CO2 condition. The
cultures of cell obtained so are called diploid cell strain. It is extremely difficult to distinguish primary cell
and the diploid cell strain. On repeated subculturing, each cell starts multiplying to form separate colony. If
each colony is removed and cultivated separately, it forms pure culture. These bunch of cells from single cell
is called clonal cell lines.
Infection with Virus - The clonal cell lines suspended in suitable media are infected with any desired virus
which replicates inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus
particles are released in the surrounding medium. These newly produced virus particles (virions) infect the
adjacent cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed.
Plaques may be detected if stained with dyes, such as neutral red or trypan blue, that can distinguish living
from dead cells. Viral growth does not always result in the lysis of cells to form a plaque. Animal viruses, in
particular, can cause microscopic or macroscopic degenerative changes or abnormalities in host cells and in
tissues called cytopathic effects, cytopathic effects may be lethal, but plaque formation from cell lysis does
not always occur.
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The recent method for cultivation and growing of Virus
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It is a homogenous collection of cells.
More convenient than animal or egg.
Prepared by treating animal tissue with enzyme to separate the individual cells.
Suspended in solution with nutrient and growth factors required by the cell
Cells grow & adhere to glass or plastic container to form a monolayer
Virus infection leads to the deterioration of cells (cytopathic effect CPE)
Total cell destruction or by diluting out count plaques and determine titer Other methods of calculating
titer: EID50, TCID50, LD50 etc
Cells isolated from human embryo, can live for hundred generation e.g. Hela cells.
Cell culture problems:
- Must be kept from microbial contamination.
- Need antibiotics for preventing contamination.
- Need experience technical worker.
- Identification of viral isolates is not easy so immunological method mainly used as depend on antibodies.
REQUIREMENTS FOR VIRAL GROWTH
All viruses are obligate intracellular parasites but they can survive in certain conditions as non-replicating
particles
 Temperature
Heat - there is great variability in the heat stability of
different viruses but icosahedral viruses tend to be relatively stable, enveloped viruses are heat labile
and most pathogenic viruses are inactivated at 55-60oC because their capsid protein is destroyed (an
important exception is the hepatitis virus.
Cold - most viruses can be preserved at sub-freezing temperatures, some can withstand lyophilisation
and can be stored in the dry state at 4oC or even at room temperature while enveloped viruses tend to
lose infectivity after prolonged storage at –90oC.
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pH
Most viruses are stable in the pH range 5-9. Enteroviruses that have to pass through the stomach can
withstand low pHs. All viruses are destroyed by alkaline conditions.
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Radiation
UV produce damaging results on double-stranded DNA that can cause inactivation of the virus. If
conditions are right the DNA can repair itself.
x-ray, gamma rays and beta particles inactivate viruses..
Stabilisation by Salts
magnesium chloride stabilises polioviruses, magnesium sulphate stabilises influenza viruses and sodium
sulphate stabilises herpes virus. Important in the preparation of vaccines e.g.. non-stabilised polio
vaccine must be stored at <0oC whereas stabilised vaccine remains potent for weeks at ambient
temperature which is an advantage when immunising in rural areas
Ether Susceptibility and Lipid Solubility
Enveloped viruses are inactivated by ether whereas non-enveloped ones are not (simple efficient test
for the presence of envelopes).
Other organic solvents and sodium deoxycholate also destroy the envelope.
Detergents
Non-ionic detergents solubilise lipid constituents but do not denature the proteins of the capsid.
Anionic detergents solubilise the lipid constituents and disrupt the capsids into separated polypeptides
 50% Glycerol
Many viruses remain alive in 50% glycerol for many years. Vaccinia virus is preserved in 50% glycerol
for many years while bacteria are killed.
 Formaldehyde
Destroys viral infectivity but has minimal adverse affect on viral antigenicity and is used in the
productions of inactivated viral vaccines
 Photodynamic Inactivation
Heterocyclic dyes like neutral red, proflavine and toluidine blue intercalate between the bases of
replicating viral
nucleic acids and on exposure to light, they become susceptible to inactivation
 Antibacterial Agents
Antibacterial antibiotics and sulphonamides have no effect on viruses although some antiviral drugs have
been developed. Oxidising agents are the most effect disinfectants e.g.. hydrogen peroxide, hypochlorite.
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2. Wite a detail account on diagnostic methods in virology
In general, diagnostic tests can be grouped into 3 categories.: (1) direct detection, (2) indirect examination (virus
isolation), and (3) serology. In direct examination, the clinical specimen is examined directly for the presence of
virus particles, virus antigen or viral nucleic acids. In indirect examination, the specimen into cell culture, eggs or
animals in an attempt to grow the virus: this is called virus isolation. Serology actually constitute by far the bulk of
the work of any virology laboratory. A serological diagnosis can be made by the detection of rising titres of
antibody between acute and convalescent stages of infection, or the detection of IgM. In general, the majority of
common viral infections can be diagnosed by serology. The specimen used for direction detection and virus
isolation is very important. A positive result from the site of disease would be of much greater diagnostic
significance than those from other sites. For example, in the case of herpes simplex encephalitis, a positive result
from the CSF or the brain would be much greater significance than a positive result from an oral ulcer, since
reactivation of oral herpes is common during times of stress.
1. Direct Examination of Specimen
1. Electron Microscopy morphology / immune electron microscopy
2. Light microscopy histological appearance - e.g. inclusion bodies
3. Antigen detection immunofluorescence, ELISA etc.
4. Molecular techniques for the direct detection of viral genomes
2. Indirect Examination
1. Cell Culture - cytopathic effect, haemadsorption, confirmation by neutralization, interference,
immunofluorescence etc.
2. Eggs pocks on CAM - haemagglutination, inclusion bodies
3. Animals disease or death confirmation by neutralization
3. Serology
Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM
in primary infection.
Classical Techniques
Newer Techniques
1. Complement fixation tests (CFT)
1. Radioimmunoassay (RIA)
2. Haemagglutination inhibition tests
2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF)
3. Particle agglutination
4. Neutralization tests
4. Western Blot (WB)
5. Single Radial Haemolysis
5. Recombinant immunoblot assay (RIBA), line immunoassay (Liatek)
1. Direct Examination
Direct examination methods are often also called rapid diagnostic methods because they can usually give a result
either within the same or the next day. This is extremely useful in cases when the clinical management of the
patient depends greatly on the rapid availability of laboratory results e.g. diagnosis of RSV infection in neonates, or
severe CMV infections in immunocompromised patients. However, it is important to realize that not all direct
examination methods are rapid, and conversely, virus isolation and serological methods may sometimes give a
rapid result. With the advent of effective antiviral chemotherapy, rapid diagnostic methods are expected to play an
increasingly important role in the diagnosis of viral infections.
1.1. Antigen Detection
Examples of antigen detection include immunofluorescence testing of nasopharyngeal aspirates for respiratory
viruses e.g.. RSV, flu A, flu B, and adenoviruses, detection of rotavirus antigen in faeces, the pp65 CMV
antigenaemia test, the detection of HSV and VZV in skin scrappings, and the detection of HBsAg in serum.
(However, the latter is usually considered as a serological test). The main advantage of these assays is that they are
rapid to perform with the result being available within a few hours. However, the technique is often tedious and
time consuming, the result difficult to read and interpret, and the sensitivity and specificity poor. The quality of the
specimen obtained is of utmost importance in order for the test to work properly.
(Virology Laboratory, Yale-New Haven Hospital)
1.2. Electron Microscopy (EM)
Virus particles are detected and identified on the basis of morphology. A magnification of around 50,000 is
normally used. EM is now mainly used for the diagnosis of viral gastroenteritis by detecting viruses in faeces e.g.
rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses. Occasionally it may be used for the detection
of viruses in vesicles and other skin lesions, such as herpesviruses and papillomaviruses. The sensitivity and
specificity of EM may be enhanced by immune electron microscopy, whereby virus specific antibody is used to
agglutinate virus particles together and thus making them easier to recognize, or to capture virus particles onto
the EM grid. The main problem with EM is the expense involved in purchasing and maintaining the facility. In
addition, the sensitivity of EM is often poor, with at least 10 5 to 106 virus particles per ml in the sample required for
visualisation. Therefore the observer must be highly skilled. With the availability of reliable antigen detection and
molecular methods for the detection of viruses associated with viral gastroenteritis, EM is becoming less and less
widely used.
Electronmicrographs of viruses commonly found in stool specimens from patients suffering from gastroenteritis.
From left to right: rotavirus, adenovirus, astroviruses, Norwalk-like viruses
1.3. Light Microscopy
Replicating virus often produce histological changes in infected cells. These changes may be characteristic or nonspecific. Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or
cytoplasm. Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies
and CMV infections respectively. Although not sensitive or specific, histology nevertheless serves as a useful
adjunct in the diagnosis of certain viral infections.
1.4.Viral Genome Detection
Methods based on the detection of viral genome are also commonly known as molecular methods. It is
often said that molecular methods is the future direction of viral diagnosis. However in practice, although the use
of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus
laboratory is still small compared to conventional methods. It is certain though that the role of molecular methods
will increase rapidly in the near future.Classical molecular techniques such as dot-blot and Southern-blot depend
on the use of specific DNA/RNA probes for hybridization. The specificity of the reaction depends on the conditions
used for hybridization. These techniques may allow for the quantification of DNA/RNA present in the specimen.
However, it is often found that the sensitivity of these techniques is not better than conventional viral diagnostic
methods.
Newer molecular techniques such as the polymerase chain reaction (PCR), ligase chain reaction (LCR),
nucleic acid based amplification (NASBA), and branched DNA (bDNA) depend on some form of amplification, either
the target nucleic acid, or the signal itself. bDNA is essentially a conventional hybridization technique with
increased sensitivity. However, it is not as sensitive as PCR and other amplification techniques. PCR is the only
amplification technique which is in common use. PCR is an extremely sensitive technique: it is possible to achieve a
sensitivity of down to 1 DNA molecule in a clinical specimen. However, PCR has many problems, the chief among
which is contamination, since only a minute amount of contamination is needed to give a false positive result. In
addition, because PCR is so sensitive compared to other techniques, a positive PCR result is often very difficult to
interpret as it does not necessarily indicate the presence of disease. This problem is particular great in the case of
latent viruses such as CMV, since latent CMV genomes may be amplified from the blood of healthy individuals.
Despite all this, PCR is being increasingly used for viral diagnosis, especially as the cost of the assay come down and
the availability of closed automated systems that could also perform quantification (Quantitative PCR) e.g. realtime PCR and Cobas Amplicor.systems. Other amplification techniques such as LCR and NASBA are just as
susceptible to contamination as PCR but that is ameliorated to a great extent by the use of propriatory closed
systems. It is unlikely though that other amplification techniques will challenge the dominance of PCR since it is
much easier to set up an house PCR assay than other assays.
2. Virus Isolation
Cell cultures, eggs, and animals may be used for isolation. However eggs and animals are difficult to handle and
most viral diagnostic laboratories depend on cell culture only. There are 3 types of cell cultures:
2.1. Types of cell cultures
1. Primary cells - e.g. Monkey Kidney. These are essentially normal cells obtained from freshly killed adult
animals. These cells can only be passaged once or twice.
2. Semi-continuous cells - e.g. Human embryonic kidney and skin fibroblasts. These are cells taken from
embryonic tissue, and may be passaged up to 50 times.
3. Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM. These are immortalized cells i.e. tumour cell
lines and may be passaged indefinitely.
Primary cell culture are widely acknowledged as the best cell culture systems available since they support the
widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply.
Continuous cells are the most easy to handle but the range of viruses supported is often limited.
2.2. Identification of growing virus
The presence of growing virus is usually detected by:
1. Cytopathic Effect (CPE) - may be specific or non-specific e.g. HSV and CMV produces a specific CPE,
whereas enteroviruses do not.
2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells. Haemadsorption is
mainly used for the detection of influenza and parainfluenzaviruses.
Confirmation of the identity of the virus may be carried out using neutralization, haemadsorption- inhibition,
immunofluorescence, or molecular tests.
Left to Right: Cytopathic effect of HSV, enterovirus 71, and RSV in cell culture. Note the ballooning of cells in the
cases of HSV and enterovirus 71. Note syncytia formation in the case of RSV.
2.3 Problems with cell culture
The main problem with cell culture is the long period (up to 4 weeks) required for a result to be available. Also, the
sensitivity is often poor and depends on many factors, such as the condition of the specimen, and the condition of
the cell sheet. Cell cultures are also very susceptible to bacterial contamination and toxic substances in the
specimen. Lastly, many viruses will not grow in cell culture at all e.g. Hepatitis B and C, Diarrhoeal viruses,
parvovirus etc.
2.4 Rapid Culture Techniques
Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. Examples
of rapid culture techniques include shell vial cultures and the CMV DEAFF test. In the CMV DEAFF test, the cell
sheet is grown on individual cover slips in a plastic bottle. After inoculation, the bottle then is spun at a low speed
for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days. The cover slip is then
taken out and examined for the presence of CMV early antigens by immunofluorescence.
Left: Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus. Also note the
presence of syncytia which is indistinguishable from that of RSV. Right: Positive CMV DEAFF test.
The role of cell culture (both conventional and rapid techniques) in the diagnosis of viral infections is being
increasingly challenged by rapid diagnostic methods i.e. antigen detection and molecular methods. Therefore, the
role of cell culture is expected to decline in future and is likely to be restricted to large central laboratories.
3. Serology
Serology forms the mainstay of viral diagnosis. This is what happens in a primary humoral immune response to
antigen. Following exposure, the first antibody to appear is IgM, which is followed by a much higher titre of IgG. In
cases of reinfection, the level of specific IgM either remain the same or rises slightly. But IgG shoots up rapidly and
far more earlier than in a primary infection. Many different types of serological tests are available. With some
assays such as EIA and RIA, one can look specifically for IgM or IgG, whereas with other assays such as CFT and HAI,
one can only detect total antibody, which comprises mainly IgG. Some of these tests are much more sensitive than
others: EIAs and radioimmunoassays are the most sensitive tests available, whereas CFT and HAI tests are not so
sensitive. Newer techniques such as EIAs offer better sensitivity, specificity and reproducibility than classical
techniques such as CFT and HAI. The sensitivity and specificity of the assays depend greatly on the antigen used.
Assays that use recombinant protein or synthetic peptide antigens tend to be more specific than those using whole
or disrupted virus particles.
3.1. Criteria for diagnosing Primary Infection
1. A significant rise in titre of IgG/total antibody between acute and convalescent sera - however, a
significant rise is very difficult to define and depends greatly on the assay used. In the case of CFT and HAI,
it is normally taken as a four-fold or greater increase in titre. The main problem is that diagnosis is usually
retrospective because by the time the convalescent serum is taken, the patient had probably recovered.
2. Presence of IgM - EIA, RIA, and IF may be are used for the detection of IgM. This offers a rapid means of
diagnosis. However, there are many problems with IgM assays, such as interference by rheumatoid factor,
re-infection by the virus, and unexplained persistence of IgM years after the primary infection.
3. Seroconversion - this is defined as changing from a previously antibody negative state to a positive state
e.g. seroconversion against HIV following a needle-stick injury, or against rubella following contact with a
known case.
4. A single high titre of IgG (or total antibody) - this is a very unreliable means of serological diagnosis since
the cut-off is very difficult to define.
3.2. Criteria for diagnosing re-infection/re-activation
It is often very difficult to differentiate re-infection/re-activation from a primary infection. Under most
circumstances, it is not important to differentiate between a primary infection and re-infection. However, it is very
important under certain situations, such as rubella infection in the first trimester of pregnancy: primary infection is
associated with a high risk of fetal damage whereas re-infection is not. In general, a sharp large rise in antibody
titres is found in re-infection whereas IgM is usually low or absent in cases of re-infection/re-activation.
3.3. Limitations of serological diagnosis
How useful a serological result is depends on the individual virus.
3.
For viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development
of antibodies. The detection of IgM or rising titres of IgG in the serum of the patient would indicate active
disease.
4. However, many viruses often produce clinical disease before the appearance of antibodies such as
respiratory and diarrhoeal viruses. So in this case, any serological diagnosis would be retrospective and
therefore will not be that useful.
5. There are also viruses which produce clinical disease months or years after seroconversion e.g. HIV and
rabies. In the case of these viruses, the mere presence of antibody is sufficient to make a definitive
diagnosis.
There are a number of problems associated with serology:1. long length of time required for diagnosis for paired acute and convalescent sera
2. mild local infections such as HSV genitalis may not produce a detectable humoral immune response
3. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and
Dengue, may lead to false positive results
4. immunocompromised patients often give a reduced or absent humoral immune response.
5. Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react
non-specifically giving a false positive result
6. Patients given blood or blood products may give a false positive result due to the transfer of antibody.
3.4. Antibody in the CSF
In a healthy person, there should be little or no antibodies in the CSF. Where there is a viral meningitis or
encephalitis, antibodies may be produced against the virus by lymphocytes in the CSF. The finding of antibodies in
the CSF is said to be significant when ratio between the titre of antibody in the serum and that in the CSF is less
than 100. But this does depend on an intact blood-brain barrier. The problem is that in many cases of meningitis
and encephalitis, the blood-brain barrier is damaged, so that antibodies in the serum can actually leak across into
the CSF. This also happens where the lumbar puncture was traumatic in which case the spinal fluid would be
bloodstained. So really, one should really check the integrity of the blood-brain barrier before making a definite
diagnosis. One way to check the integrity of the blood brain barrier is to use a surrogate antibody that most
individuals would have, such as measles virus, since most people would have been vaccinated. So the patient's
serum and CSF for measles antibody. If the blood-brain barrier is intact, there should be little or no measles
antibodies in the CSF.
UNIT V
Introduction to Nanotechnology – Scientific revolutions – Types of nanotechnology and
Nanomachines. Definition of Nanosystem- dimensionality and size dependant phenomenon;
Quantum Dots, Nanowires and Nanotubes, 2D films
--------------------------------------------------------------------------------------------------------------------Part B
1. Write a detail account on nanomachines
A nanomachine, also called a nanite, is a mechanical or electromechanical device whose dimensions are
measured in nanometers (millionths of a millimeter, or units of 10 -9 meter).
Nanomachines are largely in the research-and-development phase, but some primitive devices have been
tested. An example is a sensor having a switch approximately 1.5 nanometers across, capable of counting specific
molecules in a chemical sample. The first useful applications of nanomachines will likely be in medical technology,
where they could be used to identify pathogens and toxins from samples
This is a representation of a nanomachine. The
of body fluid. Another potential application is the detection of toxic
colored balls represent the individual atoms
chemicals, and the measurement of their concentrations, in the
that comprise the machine
environment.
The microscopic size of nanomachines translates into high
operational speed. This is a result of the natural tendency of all
machines and systems to work faster as their size decreases.
Nanomachines could be programmed to replicate themselves, or to
work synergistically to build larger machines or to construct nanochips.
Specialized nanomachines called nanorobots might be designed not only
to diagnose, but to treat, disease conditions, perhaps by seeking out
invading bacteria and viruses and destroying them.
Another advantage of nanomachines is that the individual units
require only a tiny amount of energy to operate. Durability is another potential asset; nanites might last for
centuries before breaking down. The main challenge lies in the methods of manufacture. It has been suggested
that some nanomachines might be grown in a manner similar to the way plants evolve from seeds.
Nanomachines are devices built from individual atoms. Some researchers believe that nanomachines will
one day be able to enter living cells to fight disease. They also hope to one day build nanomachines that will be
able to rearrange atoms in order to construct new objects. If they succeed, nanomachines could be used to literally
turn dirt into food and perhaps eliminate poverty.
Another goal of nanotechnology is to design nanomachines that can make copies of themselves. The
thought is that if a machine can rearrange atoms in order to build new materials, it should also be able to build
copies of itself. If this goal is achieved, products produced by nanomachines will be extremely inexpensive. This is
because the technology (once perfected) will be self-replicating and will not require specific materials, which might
be rare and therefore cost money. Arthur C. Clarke has predicted that nanotechnology will herald an end to
conventional monetary systems.
Food shortages and starvation will be a thing of the past if nanotechnology is perfected. Nanomachines
will be able to turn any material into food, and this food could be used to feed millions of people world wide.
Again, since the technology is self replicating, food produced by nanomachines will be low cost and available to all.
As well as food, nanomachines will be able to build other items to satisfy the demands of our growing population
of consumers. Clothing, houses, cars, televisions, and computers will be readily available at virtually no cost.
Furthermore, there will be no concern about the garbage produced by the new consumerist society because
nanomachines will convert it all back into new consumable goods.
Environmental problems such as ozone depletion and global warming could be solved with
nanotechnology. Swarms of nanomachines could be released into the upper atmosphere. Once there, they could
systematically destroy the ozone depleting chlorofluorocarbons (CFCs) and build new ozone molecules out of
water (H2O) and carbon dioxide (CO2). Ozone (O3) is built out of 3 oxygen atoms, and since water and
carbondioxide both contain oxygen, the atmosphere contains a plentiful supply of oxygen atoms. While the ozone
construction teams are at work in the upper atmosphere, teams of specialized nanomachines could be employed
to destroy the excess CO2 in the lower atmosphere. CO2 is a heat trapping gas, which has been identified as one of
the major contributors to global warming. Removing excess CO2 could help halt global warming and bring the
planet's ecosystem back into balance. This will benefit all species on Earth.
2. Write a detail account on Carbon Nanotubes
Carbon Nanotubes -- tiny tubes about 10,000 times thinner than a human hair -- consist
of rolled up sheets of carbon hexagons. Discovered in 1991 by researchers at NEC, they
have the potential for use as minuscule wires or in ultrasmall electronic devices. To build
those devices, scientists must be able to manipulate the Nanotubes in a controlled way.
Nanotubes and their Applications
 The properties of nanotubes have caused researchers and companies to consider
using them in several fields. For example, because carbon nanotubes have the highest
strength to weight ratio of any known material, researchers at NASA are combining
carbon nanotubes with other materials into composites as shown in the photo below
that can be used to build lightweight spacecraft.
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Another property of nanotubes is that they can easily penetrate membrances such as
cell walls. In fact, nanotubes long, narrow shape make them look like miniature
needles, so it makes sense that they can function like a needle at the cellular level.
Medical researchers are using this property by attaching molecules that are attracted
to cancer cells to nanotubes to deliver drugs directly to diseased cells.
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Another interesting property of carbon nanotubes is that their electrical resistance
changes significantly when other molecules attach themselves to the carbon atoms. Companies are using this
property to develop sensors that can detect chemical vapors such as carbon monoxide or biological molecules
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These are just a few of the potential uses of carbon nanotubes The following survey of carbon nanotube
applications introduces these and many other uses.
A survey of carbon nanotube applications under development
 Researchers and companies are working to use carbon nanotubes in various fields. The list below introduces
many of these uses. Click on any of the links below to go to a detailed explanation.
 Nanotubes bound to an antibody that is produced by chickens have been shown to be useful in lab tests to
destroy breast cancer tumors. The antibody carrying nanotubes are attracted to proteins produced by a one
type of breast cancer cell. Then the nanotubes absorb light from an infrared laser, incinerating the nanotubes
and the tumor they are attached to.
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Lightweight windmill blades made with an epoxy containing carbon nanotubes. The strength and low weight
provided by the use of nanotube filled epoxy allows longer windmill blades to be used. This increases the
amount of electricity generated by each windmill.
Aircraft using carbon nanotubes to increase strength and flexibility in highly stressed components.
Nanotube electodes in thermocells that generate electricity from waste heat.
Inexpensive nanotube based sensor that detects bacteria in drinking water. Antibodies sensitive to the
particular bacteria are bound to the nanotubes, which are then deposited onto a paper strip. When the
bacteria is present it attaches to the antibodies, changing the spacing between the nanotubes and the
resistance of the paper strip containing the nanotubes.
Combining carbon nanotubes, bucky-balls and polymers to produce inexpensive solar cells that can be formed
by simply painting a surface.
A lightweight, low power anti-icing system using carbon nanotubes in a layer coated onto aircraft wing
surfaces.
Using gold tipped carbon nanotubes to trap oil drops polluting water.
Using nanotubes as a cellular scale needle to deliver quantum dots and proteins into cancer cells.
Part C
1. Write a detail account on quantum dots and its applications
A quantum dot is a semiconductor nanostructure that confines the motion of conduction band electrons,
valence band holes, or excitons (bound pairs of conduction band electrons and valence band holes) in all three
spatial directions. The confinement can be due to electrostatic potentials (generated by external electrodes,
doping, strain, impurities), the presence of an interface between different semiconductor materials (e.g. in coreshell nanocrystal systems), the presence of the
semiconductor surface (e.g. semiconductor nanocrystal),
or a combination of these. A quantum dot has a discrete
quantized energy spectrum. The corresponding wave
functions are spatially localized within the quantum dot,
but extend over many periods of the crystal lattice. A
quantum dot contains a small finite number (of the order
of 1-100) of conduction band electrons, valence band
holes, or excitons, i.e., a finite number of elementary
electric charges.
After their discovery, semiconductor quantum
dots are emerging as a bona fide industry with a few startup companies poised to introduce products this year.
Initially targeted at biotechnology applications, such as biological reagents and cellular imaging, quantum dots are
being eyed by producers for eventual use in light-emitting diodes (LEDs), lasers, and telecommunication devices
such as optical amplifiers and waveguides. Sometimes called artificial atoms, quantum dots fall into the category of
nanocrystals, which include quantum rods and nanowires. They are technically defined as small semiconductor
crystals containing a variable number of electrons that occupy well-defined, discrete quantum states
Application
Pharmaceutical field
In the pharmaceutical domain, for example, liposomes, polymer based micro and nanoparticles have been subjects
of intense research and development during the last three decades. In this scenario metallic particles, which use
was already suggested in the first half of the '80, are now experiencing a real renaissance. In the field of diagnosis,
magnetic resonance imaging is one of the first and up to now the most developed application of metallic particles.
But beside this application, a very new generation of biosensors based on the optical properties of colloidal gold
and fluorescent nanocrystals, called quantum dots seems to be ready to be implemented in diagnosis and medical
imaging. Concerning therapeutic applications, the potentialities of metal nanoparticles to help fulfilling the need of
time and space controlled release of drugs has been intuited for a long time. It should also be used for the
detection of active ingredients with fluorescence.
In-vivo Imaging with quantum dots
Non-targeted near infrared emitting quantum dot core T2-MP EviTags were tested in tumor bearing mice. Optical
image was acquired after intravenous injection of 100pmol of T2-MP EviTags (left) or of physiological buffer as a
control (right) into the tail vein of tumor bearing mice. In this preliminary experiment, T2-MP EviTags were shown
to be capable of generating a reasonable signal to noise image when compared to the control. Further, the
biodistribution pattern as determined from the optical image shows favorable clearance of the non-targeted T2MP EviTags through the lymphatics, kidneys and bladder. No uptake in the tumor was observed, suggesting the
next round of imaging to be done with tumor targeted T2-MP EviTags will have minimal background signal within
the tumor. The development of T2-MP EviTags as non-invasive optical molecular imaging probes will have a great
impact on the early detection, diagnosis and treatment monitoring of cancer. The following image demonstrates
the ability of InGaP EviTags quantum dots to be imaged in-vivo after subcutaneous injection into a mouse liver and
tumor.
Immunoassay
An immunoassay readout method based on fluorescent imaging analysis with laser confocal scanning is described.
The ZnS-coated CdSe quantum dots (ZnS/CdSe QDs) were linked to a detection antibody. Immunoassay was carried
out on a glass chip using a sandwich assay approach, where antibody covalently bound to a glass chip was allowed
to capture antigen specially. Afterwards, the detection antibody labeled with QD was allowed to bind selectively to
the captured antigen. The fluorescent signals of the sandwich conjugate were detected by a laser confocal scanner.
A diode laser was used to excite efficiently the fluorescent signals while bovine serum albumin was used to
eliminate nonspecific binding sites. The detection limit of this approach was up to 10#8722;9 M under current
experimental conditions. The specificity of the QDs-labeled immunoglobulin (IgG) was tested by an experiment
using goat IgG and human IgG samples. The result was consistent with the binding specificity in a sandwich-type
assay.
Bimodal Molecular Imaging
The synthesis of quantum dots with a water-soluble and paramagnetic micellular coating as a molecular imaging
probe for both fluorescence microscopy and MRI. The quantum dots preserve their optical properties and have a
very high relaxivity. Targeting ligands can be coupled to these pQDs via maleimide or other functional groups. In
this study, the paramagnetic quantum dots were functionalized by conjugating them with cyclic RGD peptides and
were successfully targeted to human endothelial cells in vitro. We infer that this nanoparticulate bimodal contrast
agent may be of great use for the detection of (tumor) angiogenesis.
Summary
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Quantum dots are tiny particles, or “nanoparticles”, of a semiconductor material, traditionally chalcogenides
(selenides or sulfides) of metals like cadmium or zinc (CdSe or ZnS, for example), which range from 2 to 10
nanometers in diameter (about the width of 50 atoms).
Because of their small size, quantum dots display unique optical and electrical properties that are different in
character to those of the corresponding bulk material. The most immediately apparent of these is the
emission of photons under excitation, which are visible to the human eye as light. Moreover, the wavelength
of these photon emissions depends not on the material from which the quantum dot is made, but its size.
The ability to precisely control the size of a quantum dot enables the manufacturer to determine the
wavelength of the emission, which in turn determines the colour of light the human eye perceives. Quantum
dots can therefore be “tuned” during production to emit any colour of light desired. The ability to control, or
“tune” the emission from the quantum dot by changing its core size is called the “size quantisation effect”.
The smaller the dot, the closer it is to the blue end of the spectrum, and the larger the dot, the closer to the
red end. Dots can even be tuned beyond visible light, into the infra-red or into the ultra-violet.