Lecture: 2

Virus Structure and Multiplication

Virus Vs. Bacteria:

• Capsid and envelope

• The protein coat surrounding the nucleic acid of a virus is called the capsid.
• The capsid is composed of subunits, capsomeres, which can be a single type
of protein or several types.
• The capsid of some viruses is enclosed by an envelope consisting of lipids,
proteins and carbohydrates.
• Some envelopes are covered with carbohydrate-protein complexes called
spikes.
Growing animal viruses in the laboratory:
• Cultivation of some animal viruses requires whole animals.
• Some animal viruses can be cultivated in embryonated eggs.
• Cell cultures are cells growing in culture media in the laboratory.
• Primary cell lines and embryonic diploid cell lines grow for a short time in
vivo.
• Continuous cell lines can be maintained in vitro indefinitely.
 Viral multiplication:
Adsorption
• The first step in infection of a host bacterial cell by a phage is
adsorption.
• The tip of the virus tail becomes attached to the cell via specific
receptor sites on the cell surface.
• Attachment is specific in that certain viruses and susceptible
bacteria have complementary molecular configurations at their
opposing receptor sites.
• In some cases, the specific receptor of the bacterium is part of the
bacterial lipopolysaccharide, although any surface structure can
function as a specific phage receptor, including flagella, pili, and
carbohydrates and proteins in the membrane or cell wall.
Penetration
• The actual penetration of phage into the host cell is mechanical.
• But it may be facilitated by localized digestion of certain cell
surface structures either by phage enzymes (e.g., lysozyme) carried
on the tail of the phage or by viral activation of host degradative
enzymes.
Transcription
• Bacterial mRNA and bacterial proteins stop being synthesized
within a few minutes after entry of phage DNA.
• Bacterial DNA is quickly degraded to small fragments and the
nucleoid region of the bacterium becomes dispersed.
• Some phage mRNA is made immediately after infection.
• The amount of phage DNA increases after a brief delay.
Assembly and release
Lytic Cycle:

Lysogenic Cycle:
Multiplication of Animal viruses
 Lecture: 4
Microbial Genetics and recombinant DNA
DNA replication
The double helix of the parental DNA separates as week bonds between
the nucleotides on opposite strands break in response to the action of
replication enzymes.
RNA and Protein synthesis:
• During transcription, the enzyme RNA polymerase synthesizes a strand of
RNA from one strand of double-stranded DNA, which serves as a template.
• RNA is synthesized from nucleotides containing the bases A, C,G and U,
which pair with the bases of the DNA strand being transcribed.
• The starting point for transcription, where RNA polymerase binds to DNA,
is the promoter; the region of DNA that is the end point of transcription is
the terminator, RNA is synthesized in the 5’ to 3’ direction.
 • Translation is the process in which the information in the nucleotide base
sequence of mRNA is used to dictate the amino acid sequence of a protein.
• The mRNA associates with ribosomes, which consist of rRNA and protein.
• Three-base segments of mRNA that specify amino acids are called codons.
• The genetic code refers to the relationship among the nucleotide base
sequence of DNA, the corresponding codons of mRNA, and the amino acids
for which the codons code.
• Most amino acids are coded for by more than one codon.

Mutation:
• A mutation is a change in the nitrogenous base sequence of DNA; that
change causes a change in the product coded for by the mutated gene.
Types of mutations:
• A base substitution occurs when one base pair in DNA is replaced with a
different base pair.
• Alterations in DNA can result in missense mutations (which cause amino
acid substitutions) or nonsense mutations (which create stop codons).
• In a frameshift mutation, one or a few base pairs are deleted or added to
DNA.
• Mutagens are agents in the environment that cause permanent changes in
DNA.
• Spontaneous mutations occur without the presence of any mutagen.

Mutagens: Mutagens are agents in the environment that cause permanent

changes in DNA.

Recombinant DNA technology:

• Closely related organisms can exchange genes in natural recombination.
• Genes can be transferred among unrelated species via laboratory
manipulation, called genetic engineering.
 • Recombinant DNA is DNA that has been artificially manipulated to
combine genes from two different sources.
• A desired gene is inserted into a DNA vector, such as plasmid or a viral
genome.
• The vector inserts the DNA into a new cell, which is grown to form a clone.
• Large quantities of the gene product can be harvested from the clone.
The role of restriction enzyme in making recombinant DNA:
 Lecture:6.A
Bacterial pathogens: Bordetella pertussis and Streptococcus
pneumoniae

Characteristics of the Disease (Bordetella pertussis):

• Whooping cough is an acute respiratory disease caused by Bordetella
pertussis, a gram-negative coccobacillus.
• B. pertussis is a nutritionally fastidious bacterium and is normally cultivated
on medium containing blood, a good source of many nutrients.
• B. pertussis is spread by aerosols or by direct contact with an infected person
or an asymptomatic carrier.
 • In the first stage of the infection, the symptoms of the disease resemble those
of the common cold.
• In the second stage of the disease, a dry cough develops, which becomes
paroxysmal and is later accompanied by excess mucus production and
vomiting.
• As the second stage progresses, the episodes of coughing can become so
severe that they may cause convulsions.
Virulence factor of Bordetella pertussis:
• One of the unique characteristics of B. pertussis is its predilection for
ciliated respiratory mucosal cells.
• Because most recent studies of B. pertussis and its adhesions have focused
on cultured mammalian cell lines that have none of the characteristic
features of ciliated cells, it is still the case that we know little about the
factors that account for this remarkable host cell specificity of the bacteria.
• B. pertussis produces a number of adhesins that mediate attachment to
cultured cells.
• The two best-studied adhesins are filamentous hemagglutinin (Fha) and
pertussis toxin (Ptx).
• Filamentous hemagglutinin is a large (220 kDa) protein that forms
filamentous structures on the cell surface.
• These filaments do not have the ordered structure characteristic of pili.
• Fha can agglutinate red blood cells, a feature that explains the hemagglutinin
part of its name.

Characteristics of S. pneumoniae:
• S. pneumoniae is a gram-positive diplococcus.
• In fact, the pneumococcus was originally called Diplococcus pneumoniae.
• This morphological trait can be useful for rapid diagnosis of pneumococcal
infections.
 • Demonstration of gram-positive diplococci in properly collected sputum
specimens is a rapid method for preliminary diagnosis of pneumococcal
pneumonia.
• Similarly, demonstration of gram-positive diplococci in spinal fluid is an
early clue suggesting pneumococcal meningitis.
• In the past, a definitive diagnosis of pneumococcal pneumonia could only be
accomplished by cultivating the bacteria and subjecting them to a battery of
phenotypic tests.
• One of these tests was cultivation of the bacteria on blood agar plates.
• Colonies of S. pneumoniae produce greenish haloes (alpha-hemolysis).
• S. pneumoniae has a thick polysaccharide capsule that covers the
peptidoglycan cell wall.
• There are over 80 distinct capsular serotypes, but most cases of
pneumococcal pneumonia are caused by only 23 of these serotypes.

Virulence factors of S. pneumoniae:

• The first step in the development of pneumonia is
colonization of the nasopharynx by S. pneumoniae.
• A hypothesis concerning the factors involved in
colonization of the nose and throat has been proposed.
• On agar plates, S. pneumoniae makes two different colony
types, a transparent colony type and an opaque colony type.
Progression of two pneumococcal disease:
Short note on pneumolysin:
• One is a cytotoxic protein called pneumolysin.
• Scientists now agree that this is an important virulent factor, and
pneumolysin is being considered as a candidate for inclusion in an improved
anti-pneumococcal vaccine.
• Pneumolysin shares amino acid homology with similar cytotoxins
(hemolysins) that are produced by Streptococcus pyogenes (streptolysin) and
Listeria monocytogenes (listeriolysin).
• Pneumolysin binds cholesterol in host cell membranes and disrupts them by
forming pores.
• It might damage host mucosal cells and could aid colonization of the airway
by adversely affecting ciliated mucosal cells, thus impairing the ability of
the host to clear bacteria trapped in mucus.
• A mutant deficient in pneumolysin production had reduced virulence in
mice.
• Also, antibodies to pneumolysin conferred partial protection in intranasally
inoculated mice.
• Antibody titer to pneumolysin rise in humans with pneumonia, indicating
that the protein is being produced by bacteria growing in the host.
• Thus, pneumolysin appears to be a virulence factor, although it is not nearly
as important as the capsule or inflammatory cell wall components.
 Lecture:6.B
Bacterial pathogens: Tuberculosis and Bacillus anthracis
Tuberculosis: Tuberculosis is caused by M. tuberculosis, an obligately
aerobic rod-shaped bacterium with a gram-positive type cell wall that has an
unusually high lipid content. The disease is spread from person to person by
aerosols.
Symptoms of TB:
• fever, coughing (often with bloody sputum), weight loss, and loss of energy.
• Progressive, irreversible lung destruction occurs, and the bacteria may
escape from the lungs and enter the bloodstream.
• M. tuberculosis can infect any area of the body, including bones, joints,
liver, spleen, gastro-intestinal tract, and brain.
• The systemic form of the disease is almost always fatal.
• TB is not normally a fast-developing disease.
• Often the patient continues to decline in health for years before finally
succumbing.
Progression of the Disease:
 Virulence factors of TB:
• Studies of M. tuberculosis virulence factors have been hindered by the fact
that the generation time of M. tuberculosis is about 18 to 24 h (which means
that it takes weeks to form a visible colony on agar medium).
• Because M. tuberculosis grows so slowly, Mycobacterium smegmatis, a
faster-growing species with a generation time of 3 h, is sometimes used as a
surrogate for M. tuberculosis.
• A key virulence property of M. tuberculosis is its ability to multiply inside
monocytes and macrophages.
• It can also invade PMNs, but macrophages, especially the activated ones, are
the key to development of the infection.
• M. tuberculosis binds directly to macrophage surface protein CR3, the
normal receptor for iC3b.
• Bacillus anthracis: The majority of people exposed to spores of B.
anthracis develop cutaneous anthrax.
• Spores enter a small cut or lesion and then germinate.
• A pustule develops with a large surrounding area of swelling (edema).
• After a few days, the center of the lesion becomes black owing to necrosis.
• This lesion is called an eschar.
Forms of Anthrax :
Short note on anthrax:
• The most serious form of anthrax is inhalation (or systemic) anthrax, which
occurs when spores are inhaled and are taken up by lung macrophages.
• Inside the macrophage, the spore germinates and the bacteria begin to
divide.
• There is no apparent lung disease, but since macrophages can migrate to
lymph nodes, the bacteria spread beyond the lungs and into the blood-
stream.
• Bacteria in the bloodstream cause septic shock.
• This is the form of anthrax that can kill a person within a few days.
• Antibiotic therapy, if started within 24h, can be effective, but the fatality rate
is still very high.
• It is the sudden onset, and the difficulty in getting antibiotics to infected
people fast enough if many people are infected, that makes anthrax attractive
to bioterrorists.
• Fortunately, anthrax is not transmitted from person to person, so the risk is
in the initial exposure of a population to the weaponized anthrax spores, not
in continued transmission.
 • The A (active) portion of the edema toxin is called edema factor.
• The A portion of lethal toxin is called lethal factor.
Lecture-11
Biotechnology of vaccines

Vaccines are mainly of three types:

• 1. Dead bacteria or inactivated viruses.
• 2. Live non-virulent or weakened (attenuated) bacteria/or viruses.
• 3: Viral fragments or bacterial molecules (subunit vaccines).
Traditional vaccine: The traditional production of vaccines has several
drawbacks. It is not possible to develop vaccines for the organisms not grown in
culture. The yield of vaccines is very low. Cell cultures are costly to maintain.
There is a danger of non-virulent organisms getting converted to virulent ones.
Vaccinations by such organisms may cause the disease itself. It is not possible to
prevent all the diseases by use of traditional vaccines, e.g., AIDS.
Purified antigen vaccines: Some improvements in traditional vaccines have been
made by isolating the antigens from the pathogenic organisms. The antigens of
bacterial cell walls (e.g., Streptococcus pneumoniae causing pneumonia), and the
endotoxins are good examples of purified vaccines. The endotoxins that do not
possess toxicity but retain immunogenicity are referred to as toxoids, e.g. toxoids
of tetanus, diphtheria, etc.

Recombinant vaccines: Recombinant DNA technology in recent years, has

become a boon to produce new generation vaccines. By this approach, some of the
limitations of traditional vaccine production could be overcome.

Types of recombinant vaccines: l. Subunit recombinant vaccines: These are the

components of the pathogenic organisms. Subunit vaccines include proteins,
peptides and DNA.

• 2. Attenuated recombinant vaccines: These are the genetically modified

pathogenic organisms (bacteria or viruses) that are made non-pathogenic and
used as vaccines.

• 3. Vector recombinant vaccines: These are the genetically modified viral

vectors that can be used as vaccines against certain pathogens.

• Hepatitis B: Hepatitis B is a widespread disease in man. It primarily affects

liver causing chronic hepatitis, cirrhosis and liver cancer. The gene encoding
for hepatitis B surface antigen (HBsAg) has been identified. Recombinant
hepatitis B vaccine as a subunit vaccine, is produced by cloning HbsAg gene
in yeast cells. Saccharomyces cerevisiae is used for this purpose. The gene
for HBsAg is inserted (pMA 56) which is linked to the alcohol
dehydrogenase promoter. These plasmids are then transferred and cultured.
Tuberculosis: Tuberculosis is caused by the bacterium Mycobacterium
tuberculosis. It is often fatal, and as per some estimates nearly 3 million deaths
occur every year due to this highly infectious disease. Antibiotics are used to treat
tuberculosis. However, drug-resistant M. tuberculosis strains have been developed
making the drug therapy sometimes ineffective. Vaccination for tuberculosis is
therefore, suggested.

AIDS (Acquired immunodeficiency syndrome): AIDS is a retroviral disease

caused by human immunodeficiency virus (HIV). This disease is characterized by
immunosuppression, neoplasma and neurological manifestations. AIDS is
invariably fatal, since as of now there is no cure. Development of a vaccine against
AIDS is a top priority by DNA technologists world over. In fact, vaccines are
being continuously developed and field tested, although there has been no success
so far.

The most important limitations of the vaccine are that the HIV has high frequency
of mutations. Therefore the vaccines developed cannot hind to the new virus (i.e.,
mutated one). addition, gp120 and gp41 are very poor stimulators of immune
system.

DNA vaccine: A DNA vaccine consists of a gene encoding an antigenic protein,

inserted onto a plasmid, and then incorporated into the cells in a target animal.
DNA vaccine-plasmids can be administered to the animals by one of the following
delivery methods.

• -Nasal spray
 • -Intramuscular injection

• -Intravenous injection

• -Intradermal injection

• -Gene gun (involves pressure delivery of DNA-coated gold beads)

Humoral immunity: As the antigen molecules bind to B-lymphocytes, they

trigger the production of antibodies which can destroy the pathogens. Some of the
B-lymphocytes become memory cells that can protect the host against future
infections.

Cellular immunity: The protein fragments of the antigen bound to MHC

molecules can activate the cytotoxic T-lymphocytes. They are capable of
destroying the infected pathogenic cells. Some of the activated T-lymphocytes
become memory cells which can kill the future infecting pathogens.

Advantages of DNA vaccines: The tedious and costly procedures of purifying

antigens or creating recombinant vaccines are not necessary.

DNA vaccines are very specific in producing the target proteins (antigens or
antibodies). Thus, they trigger immune response only against the specific
pathogen.

• In general, DNA vaccines elicit much higher immune response compared to

other kinds of vaccines.

• DNA vaccines are more stable for temperature variations (low or high) than
the conventional vaccines. Thus, the storage and transport problems
associated with vaccines are minimal.

• The delivery methods to the host are simpler for DNA vaccines.
Disadvantages of DNA vaccines:

• 1. The fate of the DNA vaccine in the host cells is not yet clear.

• There is a possibility of this DNA getting integrated into the host genome
and this may interrupt the normal functions.

• 2. There also exists a danger of cancer due to DNA vaccines.

• 3. The post-translational modification of the gene (DNA vaccine) product in

host cells may not be the same as that found in the native antigen.

• Edible vaccine production and use: The bacterium, Agrobacterium

tumefaciens is commonly used to deliver the DNA (genetic material) for
bacterial or viral antigens.

• A plasmid carrying the antigen gene and an antibiotic resistance gene are
incorporated into the bacterial cells (A. tumefaciens).

• The cut pieces of potato leaves are exposed to an antibiotic which can kill
the cells that lack the new genes.

• The production of vaccine potatoes is illustrated in Fig.

Attenuated recombinant vaccines: In the early years of vaccine research,
attenuated strains of some pathogenic organisms were prepared by prolonged
cultivation - weeks, months or even years. Although the reasons are not known, the
infectious organism would lose its ability to cause disease but retains its capability
to act as an immunizing agent. This type of approach is almost outdated now. It is
now possible to genetically engineer the organisms (bacteria or viruses) and use
them as live vaccines, and such vaccines are referred to as attenuated recombinant
vaccines.

The genetic manipulations for the production of these vaccines are broadly of
two types:

l. Deletion or modification of virulence genes of pathogenic organisms.

2. Genetic manipulation of non-pathogenic organisms to carry and express antigen

determinants from pathogenic organisms.
Cholera: Cholera is an intestinal disease characterized by diarrhea, dehydration,
abdominal pain and fever. It is caused by the bacterium, Vibrio cholerae. This
pathogenic organism is transmitted by drinking water contaminated with fecal
matter. Cholera epidemics are frequently seen in developing countries where the
water purification and sewage disposal systems are not well developed. On
entering the small intestine, V. cholerae colonizes and starts producing large
amounts of a toxic protein, a hexameric enterotoxin. This enterotoxin stimulates
the cells lining intestinal walls to release sodium, bicarbonate and other ions.

1. A tetracycline resistance gene was inserted into the A1 peptide sequence of

V. cholerae chromosome. This destroys the DNA sequence encoding for A1
peptide, besides making the strain resistant to tetracycline. Unfortunately,
 the tetracycline resistant gene is easily lost and the enterotoxin activity is
restored. Because of this, the new strain of V. cholerae as such cannot be
used as a vaccine.
2. The DNA sequence of A1 peptide is incorporated into a plasmid, cloned and
digested with restriction enzymes. In this manner, the A1 peptide coding
sequence is deleted. By using T4 DNA ligase, the plasmid is re-circularized.
This plasmid contains a small portion of A1 peptide coding sequence.
3. The plasmid, containing the deleted A1 peptide sequence is transferred by
conjugation into the V. cholerae strain carrying a tetracycline resistance
gene.
4. Recombination can occur between the plasmid and the chromosome of V.
cholera. The result of this double crossover is the formation of V. cholerae
containing a chromosomal DNA lacking A1 peptide DNA sequence.
As the bacterium, V. cholerae multiplies, the plasmids are lost in the next
few generations.
5. The V. cholerae cells defective in A1 peptide are selected, based on
tetracycline sensitivity. It may be noted that this new strain lacks tetracycline
resistance gene.