MOLECULAR BIOLOGY

OF TROPICAL INFECTIOUS DISEASES

dr. Tri Wibawa, PhD

Department of Microbiology
Gadjah Mada Sch. of. Med

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Eukaryote versus prokaryote

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Watson and Cricks model for DNA replication

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Eukaryotes genomes;
Divided into two or more linear DNA molecules.
Each contained in a different chromosome.
Possess smaller, usually circular, mitochondrial
genomes.
Third genome located in the chloroplast (in plant and
other photosynthetic organism)
10 Mb 100,000 Mb in length.
Higher eukaryotes need larger genome to
accommodate the extra genes.
Correlation between genome size and complexity (???)
--- C-value paradox.
Space is saved in the gnomes of less complex
organism because the genes are more closely packed
together.
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Prokaryotes genomes:
Whole prokaryotes genomes are smaller than
eukaryotes.
Most if not all -- are contained in a single DNA
molecules.
The molecule is circular.
Have second circular or linear genome, called
PLASMID.
Have fewer genes
More compact genome organization, more gene but
less space.
There is NO INTRONS (some exception is in
archaea.)
Infrequency of repetitive sequences.
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upstream

exon intron exon

Initiation codon

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downstream

termination codon

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Circular shape of microbial DNA

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Eukaryotes, Prokaryotes,
and Virus Size

More
than 500 viruses could fit inside
a single bacterial cell.
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Virion Structure
Lipid Envelope

Nucleic Acid

Protein
Capsid
Virion
Associated
Polymerase

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Spike
Projections
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VIRAL
SHAPE
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Helical Viruses

Rabies Virus

Measles Virus

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Icosahedron

Herpes virus
parvovirus
Icosahedron polyhedron
with 20 triangular faces

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poliovirus

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Complex

smallpox

Influenza
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virus

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Bacteriophage

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Components of Viruses

1.

Genome
nucleic acid core of the virus
Consists of either DNA or RNA

2.

Capsid
outer protein coat of the virus
Formed from smaller protein units called capsomeres

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Components of Viruses

3. Envelope
Enclosing structure, similar to the membrane that
encloses a cell
Acquired at last stage of replication

4. Spikes
Projections from envelope
Helps virus contact host cell

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VIRUS GENOMES
Is more varied than any other kingdoms
Single / double strand
Linear / circular / segmented
Single strand virus genome may be:
Positive (+) sense (the same polarity as mRNA)
Negative (-) sense (need virus-specific polymerase)
ambisense (mixture of the two)

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(+) and (-) Sense

(+) RNA and (+) DNA have the same sequence
as the mRNA (except that in DNA thymine
replaces uracil).
() RNA and () DNA have the sequence
complementary to the mRNA (except that in
DNA thymine replaces uracil).
Most of these viruses have either a (+) or a ()
strand genome.
Some ssDNA viruses and some ssRNA viruses have
ambisense genomes.
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Replication of DNA Genomes

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Replication of RNA Genomes

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Reverse-Transcribing
Viruses

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Locations of virus genome

replication in eukaryotic cells

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Locations of virus genome

replication in eukaryotic cells

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Infection

Transfection
Purified
Nucleic acids
(DNA or (+)RNA)

Infectious
Virus particle

Host cells

Infectious virus particle

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Infectious virus particle

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Influenza virus

Segmented
8 segments
negative-sense RNA

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Gemini virus

Bipartite
Two molecules
Ds-DNA

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DNA viruses

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RNA viruses

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Replication of viruses :

Attachment, penetration and un-coating

Expression of viral genome
Synthesis of viral component
Morphogenesis and release

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Semliki forest virus

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Obligate intracellular parasites

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Proses
replikasi
Virus Influenza

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Peran HA
pada proses
infeksi
Segmen
genome
berada di
dalam sel
host naked
& free

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Pathogenic fungal genera grouped by taxonomic class

(the most common genera in bold type)
Basidiomycetes
Z
y
g
o
m
y
c
e
t
e
s

Cryptococcus
Malassezia
Mucor
Rhizopus
Absidia
Saksenaea
Cunninghamella
Entomophthora
Basidiobolus

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MAN
Candida
Trichosporon
Acremonium
Exophiala
Bipolaris
Deuteromycetes
(fungi imperfecti)

Histoplasma
Blastomyces
Aspergillus
Trichophyton
Epidermophyton
Microsporum
Sporothrix
Pseudallescheria

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A
s
c
o
m
y
c
e
t
e
s

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BIOLOGY OF THE FUNGI

Somatic structures:
Mold form
Yeast form
Dimorphic

Mold form

Yeast form

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M. tuberculosis

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Molecular basis of antimicrobial resistance

Mechanisms of antimicrobial resistance:
The presence of inactivation enzyme
Alternative protein for the target of antimicrobial agent
Mutation in the target
Post-transciptional and post-translational modification
of the target.
Reduction of the antimicrobial uptake.
Active efflux of antimicrobial agent.
Over-expression of the target.
Unknown mechanism

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Rifampisin (RIF)
Action: Inhibition of DNA-dependent RNA polymerase.
RNA polymerase: 4 subunit: ; ; ;
Genes : rpoA; rpoB; rpoC; rpoD
Target: bind to the subunit resulting in transcription
inhibition
Mutation in the rpoB gene responsible to rifampisin
resistance.

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Identified mutations in rpoB genes of MTB

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Mutation in more than one

residue of the rpoB gene.

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Isoniazid (INH)
Action and target: Not clearly known
Candidate; INH or INH metabolite block the synthesis
of mycolic acids.
Genes : katG. Encoding catalase-peroxidase enzym
INH resistance MTB had decreased catalase activity.
Mutation in the katG gene responsible to isoniazid
resistance.

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Isoniazid (INH)
Genes : inhA. Encoding protein for fatty acid
biosynthesis.
inhA has correlation with resistance to INH and ETH
Polymorphisms were found in the upstream of orfI

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Ethambutol (EMB)
Action and target: Not clearly known
Candidate;
Inhibition of RNA metabolism
Inhibition of phospholipid synthesis
Inhibition of transfer of mycolic acid
Inhibition of spermidine synthesis
Inhibition of first step of glucose conversion.
Genes : No genes were identified.

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Pyrazinamide (PZA)
Action and target: Not clearly known
Candidate; Pyrazinamidase convert PZA to pyarzinoic
acid. PZA-resistance MTB lack of the pyrazinamidase
activity.
Genes : No gene were identified

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Molecular Diagnostic Techniques of

Tuberculosis
Molecular Diagnostic Techniques are indicated:
Detection of organisms that cannot be grown in vitro or for
which current culture techniques are too insensitive.
Require complex media and prolong incubation time.
Molecular Diagnostic Techniques consideration:
high sensitivity
high specificity
speed
simplicity
clinical relevance.
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Basic principle of any Molecular Diagnostic

Techniques is the detection of specific nucleic
acid (DNA) sequence of the pathogens.
Example:
Polymerase chain reaction (PCR)
Southern blot hybridization
Nested PCR
Multiplex PCR
Reverse transcriptase PCR (RT PCR)
Trancription-mediated amplification (TMA)
Ligase chain reaction (LCR)
Nucleic acid sequence-based amplification (NASBA)
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PCR versus Conventional diagnostic

procedures
At present: PCR cannot replace the conventional
Reason:
Sensitivity
Specificity
Specimen still need to be culture for susceptibility test.
Cost

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Avian influenza

Timeline of Emergence of
Influenza A Viruses in Humans
Avian
Influenza
Russian
Influenza

H9
H5

H7
H5

H1
H3
H2

H1
1918
Spanish
Influenza
H1N1

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1957 1968 1977

Asian
Hong
Influenza Kong
H2N2 Influenza
H3N2 tri wibawa

1997 2003
1998/9
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Antigenic Drift
Antigenic drift is the natural mutation
over time of known strains of influenza
to evade the immune system.
Antigenic drift occurs in all types of
influenza including influenza A, B and
C.
The same subtype of viruses

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MUTASI VIRUS INFLUENZA

Virus Influenza tidak memiliki mekanisme:
Proofreading
Repair of errors yang terjadi saat replikasi
Akibat tidak adanya koreksi thd kesalahan :
Komposisi genetik dari virus berubah pada saat
bereplikasi pada host.
Strain yang beredar berganti dengan varian virus baru
dengan sifat antigenik yang berbeda.
Perubahan yang biasanya kecil ini konstan,

permanet dan biasanya kecil ini merubah

komposisi antigenik virus influenz A.
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Mutation
In general mutations are changes to the DNA or RNA
(genetic materials) of viruses.
Mutations can be caused by :

Copying errors in the genetic material during cell division

Exposure to elements such as radiation, chemicals, viruses
or can occur deliberately under cellular control during the
processes such as meiosis or hyper-mutation.

Mutations are considered the driving force of

evolution
Less favorable (or deleterious) mutations are
removed from the gene pool by natural selection.
Favorable (beneficial or advantageous) ones tend to
accumulate.
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Mutation (in humans)

Migratory
water birds

Source: WHO/WPRO

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Influenza Surface Proteins

Neuraminidase

Hemagglutinin

RNA

M2 protein
(only on type A)

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Antigenic Drift

RNA
Hemagglutinin
Neuraminidase
Antibodies
Sialic
acid
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Antigenic Shift:
Antigenic shift is the process by which two different
strains of influenza combine to form a new subtype
having a mixture of the surface antigens of the two
original strains.
The term antigenic shift is specific to the influenza
literature; in other viral systems, the same process
is called reassortment or viral shift.
Antigenic shift occurs only in influenza A because it
infects more than just humans.
Affected species include other mammals and birds,
giving influenza A the opportunity for a major
reorganization of surface antigens.
New subtype of virus developed
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Reassortment

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Reassortment (in humans)

Migratory
water birds

Source: WHO/WPRO

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Reassortment (in pigs)

Migratory
water birds

Domestic bird

Source: WHO/WPRO

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From birds to humans

Migratory
water birds

Domestic birds

Hong Kong,
SAR China 1997,
H5N1
Hong Kong,
SAR China 1999,
H9N2
The
Netherlands
2003, H7N7
Hong Kong,
SAR China 2003,
H5N1

Source: WHO/WPRO

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Antigenic Shift

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