SBT 703 Microbiology

Dr.Akhileshwar Namani Ph.D

 Units of Measurement
When measuring microorganisms, we use the metric system.

A major advantage of the metric system is that units relate to each other by factors
of 10.

Thus, 1 meter (m) equals 10 decimeters (dm) or 100 centimeters (cm) or 1000
millimeters (mm).

Units in the U.S. system of measure do not have the advantage of easy conversion
by a single factor of 10. For example, 3 feet, or 36 inches, equals 1 yard.

Microorganisms are measured in even smaller units, such as micrometers and

nanometers.

A micrometer (µm) equals 0.000001 m (10-6 m). The prefix micro indicates the
unit following it should be divided by 1 million, or 106.

A nanometer (nm) equals 0.000000001 m (10-9 m). Angstrom (Å) was

previously used for 10-10 m, or 0.1 nm
 Microscopy
The microscope is the microbiologist’s oldest and most fundamental tool for
studying microorganisms.

Leeuwenhoek’s microscope was a light microscope, and his design used a simple
lens that could magnify an image at least 266 times.

The simple microscope used by van Leeuwenhoek in the seventeenth century had
only one lens and was similar to a magnifying glass.

However, van Leeuwenhoek was the best lens grinder in the world in his day. His
lenses were ground with such precision that a single lens could magnify a microbe
300x.

His simple microscopes enabled him to be the first person to see bacteria

Contemporaries of van Leeuwenhoek, such as Robert Hooke, built compound

microscopes, which have multiple lenses.

In fact, a Dutch spectacle maker, Zaccharias Janssen, is credited with making the
first compound microscope around 1600 .
 Microscopy

However, these early compound microscopes were of poor quality and could not be
used to see bacteria.

It was not until about 1830 that a significantly better microscope was developed by
Joseph Jackson Lister (the father of Joseph Lister).

Various improvements to Lister’s microscope resulted in the development of the

modern compound microscope, the kind used in microbiology laboratories today.

Microscopic studies of live specimens have revealed dramatic interactions between

microbes
 Microscopy

In a light microscope the sample is illuminated with visible light

Magnification
Resolution

Magnification describes the capacity of a microscope to enlarge an image. All

microscopes employ lenses that provide magnification. Magnification, however, is
not the limiting factor in our ability to see small objects. It is resolution that governs
our ability to see the very small

Resolution is the ability to distinguish two adjacent objects as distinct and

separate. The limit of resolution for a light microscope is about 0.2 μm (μm is the
abbreviation for micrometer, 10-6 m). What this means is that two objects that are
closer together than 0.2 μm cannot be resolved as distinct and separate.
 Microscopy
Microscopy has improved considerably since the days of Leeuwenhoek.

Several types of light microscopy are now available, including

bright-field,
phase-contrast,
differential interference contrast,
dark-field, and fluorescence.

With the modern compound light

microscope, light from a light source is focused
on the specimen by the condenser, and this
light passes through the sample and is
collected by the lenses.

The modern compound light microscope

contains two types of lenses, objective and
ocular, that function in combination to
magnify the image.
 Microscopy

Microscopes used in microbiology have ocular

lenses that magnify 10–30X and objective
lenses that magnify 10–100X).

The total magnification of a compound light

microscope is the product of the magnification
of its objective and ocular lenses.

Magnification of 1000X is required to

resolve objects 0.2 μm in diameter, which is the
limit of resolution for most light microscopes
(increasing magnification beyond 1000X
provides little improvement in the resolution of
a light microscope).

The limit of resolution for a light microscope

is a function of the wavelength of light used and
the light-gathering ability of the
objective lens, a property known as its
numerical aperture
 Microscopy

There is a correlation between the magnification of a lens and its numerical

aperture; lenses with higher magnification typically have higher numerical apertures.

The diameter of the smallest object resolvable by any lens is equal to 0.5
 λ/numerical aperture, where  λ is the wavelength of light used.

With objectives that have a very high numerical aperture (such as the
100x objective), an optical grade oil is placed between the microscope slide
and the objective.

Lenses on which oil is used are called oil-immersion lenses.

Immersion oil increases the light gathering ability of a lens, that is, it increases the
amount of light that is collected and viewed by the lens

Immersion oils are transparent oils that have specific optical

and viscosity characteristics necessary for use in microscopy. 
 Microscopy

In light microscopy, oil immersion is a technique used to increase the resolving

power of a microscope.

Resolving power is the ability of an imaging device to separate (i.e., to see as

distinct) points of an object that are located at a small angular distance or it is the
power of an optical instrument to separate far away objects, that are close together,
into individual images

The term resolution or minimum resolvable distance is the minimum distance

between distinguishable objects in an image, although the term is loosely used by
many users of microscopes and telescopes to describe resolving power.

Angular distance  is the angle between the two sightlines, or between two point
objects as viewed from an observer.

Before the development of synthetic immersion oils in the 1940s, cedar tree oil was
widely used
 Microscopy

Type A and Type B are both general purpose immersion oils with different
viscosities.

 Type F immersion oil is best used for fluorescent imaging at room temperature
(23 °C), while type N oil is made to be used at body temperature (37 °C) for live cell
imaging applications.

The physical properties of the medium

through which light rays travel
determines the degree to which the
light will be refracted. The ‘refractive
index’ is a numerical value (without
units) which is a determinant of the
extent to which light will refract when
passing through a material.
 Microscopy

In light microscopy, specimens are visualized because of differences in contrast that
exist between them and their surroundings.

In brightfield microscopy, contrast results when cells absorb or scatter light
differently from their surroundings.

Bacterial cells typically lack contrast, that is, their optical properties are similar to
the surrounding medium, and hence they are difficult to see well with the bright-field
microscope.

Pigmented microorganisms are an exception because the color of the organism adds
contrast, thus improving visualization by bright-field optics. For cells lacking
pigments there are several ways to boost contrast, and we consider these methods in
the next section.

Cells can be stained to improve contrast, and staining is commonly used to visualize
bacteria with bright-field microscopy.
 Microscopy

In addition to staining, other methods of light microscopy

have been developed to improve contrast, such as

phase contrast,

differential interference contrast,

dark field, and fluorescence

 Dark field Microscopy
It is not always desirable to stain a specimen, but an unstained cell has little
contrast with its surroundings and is therefore difficult to see. Unstained cells are
more easily observed with the modified compound microscopes
A darkfield microscope is used to examine live microorganisms that either are
invisible in the ordinary light microscope, cannot be stained by standard methods,
or are so distorted by staining that their characteristics are unclear.
A darkfield microscope uses a darkfield condenser that contains an opaque
disk. (Non transparent, objects which doesn't leave light to pass through it).
The disk blocks light that would enter the objective lens directly.
Only light that is reflected off (turned away from) the specimen enters the
objective lens.
Because there is no direct background light, the specimen appears light against a
black background—the dark field .
This technique is frequently used to examine unstained microorganisms
suspended
in liquid.
One use for darkfield microscopy is the examination of very thin spirochetes, such
as Treponema pallidum , the causative agent of syphilis
(a) Bright field. The path of light in (b) Darkfield. The darkfield microscope
bright field microscopy, the type of uses a special condenser with an opaque
illumination produced by regular disk that eliminates all light in the center
compound light microscopes. of the beam. The only light that reaches
the specimen comes in at an angle; thus,
only light reflected by the specimen (blue
lines) reaches the objective lens
 Comparison

Brightfield illumination shows internal Against the black background seen with
structures and the outline of the transparent darkfield microscopy, edges of the cell are
pellicle (external covering ) bright, some internal structures seem to
sparkle, and the pellicle is almost visible.
 Phase-contrast Microscopy
A phase-contrast microscope converts slight differences in refractive index and
cell density into easily detected variations in light intensity
Phase-contrast microscopy is especially useful to examine the internal
structures of a cells of living microorganisms.
In addition, it isn’t necessary to fix (attach the microbes to the microscope
slide) or stain the specimen -procedures that could distort or kill the
microorganisms.
In a phase-contrast microscope, one set of light rays comes directly from the
light source. The other set comes from light that is reflected or diffracted from a
particular structure in the specimen.
(Diffraction is the scattering of light rays as they “touch” a specimen’s edge.
The diffracted rays are bent away from the parallel light rays that pass farther
from the specimen.)
When the two sets of light rays—direct rays and reflected or diffracted rays are
brought together, they form an image of the specimen on the ocular lens,
containing areas that are relatively light (in phase), through shades of gray, to
black (out of phase)
 Phase-contrast Microscopy
Phase-contrast. (Top) In phase-
contrast microscopy, the specimen is
illuminated by light passing through an
annular (ringshaped) diaphragm.
Direct light rays (unaltered by the
specimen) travel a different path from
light rays that are reflected or
diffracted as they pass through the
specimen. These two sets of rays are
combined at the eye. Reflected or
diffracted light rays are indicated in
blue; direct rays are red. (Bottom)
Phase-contrast microscopy shows
greater differentiation of internal
structures and clearly shows the
pellicle
 Fluorescence Microscopy
The light microscopes thus far considered produce an image from light that
passes through a specimen. An object also can be seen because it emits light:
this is the basis of fluorescence microscopy
Fluorescence microscopy takes advantage of fluorescence, the ability of
substances to absorb short wavelengths of light (ultraviolet) and give off light
at a longer wavelength (visible).
Some organisms fluoresce naturally under ultraviolet light; if the specimen
to be viewed does not naturally fluoresce, it is stained with one of a group of
fluorescent dyes called fluorochromes.
When microorganisms stained with a fluorochrome are examined under a
fluorescence microscope with an ultraviolet or near-ultraviolet light source,
they appear as luminescent, bright objects against a dark background

Commonly Used Fluorochromes

 Fluorescence Microscopy
Fluorochromes have special attractions for different microorganisms. For
example, the fluorochrome auramine O, which glows yellow when exposed to
ultraviolet light, is strongly absorbed by Mycobacterium tuberculosis.
When the dye is applied to a sample of material suspected of containing the
bacterium, the bacterium can be detected by the appearance of bright yellow
organisms against a dark background.
Bacillus anthracis, the causative agent of anthrax, appears apple green when
stained with another fluorochrome, fluorescein isothiocyanate (FITC)
The most commonly used fluorescence microscopy is epifluorescence
microscopy, also called incident light or reflected light fluorescence microscopy.
Epifluorescence microscopes employ an objective lens that also acts as a
condenser mercury vapor arc lamp or other source produces an intense beam of
light that passes through an exciter filter.
The exciter filter transmits only the desired wavelength of light.
The excitation light is directed down the microscope by the dichromatic mirror
This mirror reflects light of shorter wavelengths (i.e., the excitation light)
but allows light of longer wavelengths to pass through
The excitation light continues down, passing through the objective lens to the
specimen, which is usually stained with molecules called fluorochromes
Fluorescence Microscopy
 Fluorescence Microscopy
The fluorochrome absorbs light energy from the excitation light and fluoresces
brightly.
The emitted fluorescent light travels up through the objective lens into the
microscope.
Because the emitted fluorescent light has a longer wavelength, it passes through
the dichromatic mirror to a barrier filter, which blocks out any residual excitation
light.
Finally, the emitted light passes through the barrier filter to the eyepieces.
The fluorescence microscope has become an essential tool in microbiology.
Bacterial pathogens can be identified after staining with fluorochromes or
specifically tagging them with fluorescently labeled antibodies using
immunofluorescence procedures.
In ecological studies, fluorescence microscopy is used to observe microorganisms
stained with fluorochrome-labeled probes or protein of interest to a gene isolated
from jellyfish belonging to the genus Aequorea.
This jellyfish gene encodes a protein that naturally fluoresces green when
exposed to light of a particular wavelength and is called green fluorescent protein
(GFP). Thus when the protein is made by the cell, it is fluorescent. GFP has been
used extensively in studies on bacterial cell division and related phenomena
 Confocal Microscopy
Confocal microscopy is a technique in light microscopy used to
reconstruct three-dimensional images.
 Like fluorescent microscopy, specimens are stained with fluorochromes
so they will emit, or return, light.
But instead of illuminating the entire field, one plane of a small region of
a specimen is illuminated with a short-wavelength (blue) light which passes
the returned light through an aperture aligned with the illuminated region.
 Each plane corresponds to an image of a fine slice that has been
physically cut from a specimen.
Successive planes and regions are illuminated until the entire specimen
has been scanned.
Because confocal microscopy uses a pinhole aperture, it eliminates
blurring that occurs with other microscopes. As a result, exceptionally clear
two-dimensional images can be obtained, with improved resolution of up
to 40% over that of other microscopes.
 Confocal Microscopy
Most confocal microscopes are used in conjunction with computers to construct
three-dimensional images.
 The scanned planes of a specimen, which resemble a stack of images, are
converted to a digital form that can be used by a computer to construct a three-
dimensional representation.
Computers are integral to the process of creating confocal images. A computer
interfaced with the confocal microscope receives digitized information from each
plane in the specimen that is examined
This information can be used to create a composite image that is very clear and
detailed or to create a threedimensional reconstruction of the specimen
The reconstructed images can be rotated and viewed in any orientation. This
technique has been used to obtain threedimensional images of entire cells and
cellular components
Confocal microscopy has numerous applications. One is the study of biofilms,
which can form on many different types of surfaces, including indwelling medical
devices such as hip joint replacements
In addition, confocal microscopy can be used to evaluate cellular physiology by
monitoring the distributions and concentrations of substances such as ATP and
calcium ions
 Confocal Microscopy

A Ray Diagram of a Confocal Microscope. The yellow lines represent laser light used for
illumination. Red lines symbolize the light arising from the plane of focus, and the blue lines
stand for light from parts of the specimen above and below the focal plane.
 Electron Microscopy
For centuries the light microscope has been the most important instrument for studying
microorganisms. However, even the best light microscopes have a resolution limit of about 0.2
micro meter, which greatly compromises their usefulness for detailed studies of many
microorganisms
 Electron Microscopy

Viruses, for example, are too small to be seen with light

microscopes.
Bacteria and archaea can be observed, but because they are usually
only 1 to 2 μm in diameter,only their general shape and major
morphological features are visible.
The detailed internal structure of larger microorganisms also cannot
be effectively studied by light microscopy.
These limitations arise from the nature of visible light waves, not
from any inadequacy of the light microscope itself.
Electron microscopes have much greater resolution. In electron
microscopy, a beam of electrons is used instead of light.
Like light, free electrons travel in waves. The resolving power of the
electron microscope is far greater than that of the other microscopes
discussed before.
 Electron Microscopy

The better resolution of electron microscopes is due to the shorter

wavelengths of electrons; the wavelengths of electrons are about
100,000 times smaller than the wavelengths of visible light.
Thus, electron microscopes are used to examine structures too small
to be resolved with light microscopes. Images produced by electron
microscopes are always black and white, but they
may be colored artificially to highlight certain details.
Instead of using glass lenses, an electron microscope uses
electromagnetic lenses to focus a beam of electrons onto a specimen.

There are two types: the transmission electron microscope and

the scanning electron microscope
 Transmission electron Microscopy
In the transmission electron microscope (TEM), a finely focused beam of
electrons from an electron gun passes through a specially prepared, ultrathin
section of the specimen
The beam is focused on a small area of the specimen by an electromagnetic
condenser lens that performs roughly the same function as the condenser of a
light microscope—directing the beam of electrons in a straight line to illuminate
the specimen.
Instead of being placed on a glass slide, as in light microscopes, the specimen is
usually placed on a copper mesh grid.
The beam of electrons passes through the specimen and then through an
electromagnetic objective lens, which magnifies the image.
Finally, the electrons are focused by an electromagnetic projector lens (rather
than by an ocular lens as in a light microscope) onto a fluorescent screen or
photographic plate.
The final image, called a transmission electron micrograph, appears as many light
and dark areas, depending on the number of electrons absorbed by different areas
of the specimen
The transmission electron microscope can resolve objects as close together as 10
pm, and objects are generally magnified 10,000 to 100,000 X.
 Transmission electron Microscopy
Because most microscopic specimens are so thin, the contrast between their
ultrastructures and the background is weak.
Contrast can be greatly enhanced by using a “dye” that absorbs electrons and
produces a darker image in the stained region.
Salts of various heavy metals, such as lead, osmium, tungsten, and uranium, are
commonly used as stains.
 These metals can be fixed onto the specimen (positive staining) or used to
increase the electron opacity (cloudiness) of the surrounding field (negative
staining).
Negative staining is useful for the study of the very smallest specimens, such as
virus particles, bacterial flagella, and protein molecules
In addition to positive and negative staining, a microbe can be viewed by a
technique called shadow casting.
In this procedure, a heavy metal such as platinum or gold is sprayed at an angle
of about 45° so that it strikes the microbe from only one side.
The metal piles up on one side of the specimen, and the uncoated area on the
opposite side of the specimen leaves a clear area behind it as a shadow.
This gives a three-dimensional effect to the specimen and provides a general
idea of the size and shape of the specimen
Transmission electron Microscopy
 Transmission electron Microscopy
Transmission electron microscopy has high resolution and is
extremely valuable for examining different layers of specimens
However, it does have certain disadvantages. Because electrons
have limited penetrating power, only a very thin section of a
specimen (about 100 nm) can be studied effectively.
Thus, the specimen has no three-dimensional aspect. In addition,
specimens must be fixed, dehydrated, and viewed under a high
vacuum to prevent electron scattering.
 These treatments not only kill the specimen, but also cause
shrinkage and distortion, sometimes to the extent that there may
appear to be additional structures in a prepared cell.
Structures that appear as a result of the preparation method are
called artifacts
Transmission electron Microscopy
 Scanning electron Microscopy
Transmission electron microscopes form an image from radiation that has
passed through a specimen. The scanning electron microscope (SEM) produces
an image from electrons released from atoms on an object's surface.
The SEM has been used to examine the surfaces of microorganisms in great
detail; many SEMs have a resolution of 7 nm or less
The scanning electron microscope (SEM) overcomes the sectioning problems
associated with a transmission electron microscope.
It provides striking three-dimensional views of specimens. An electron gun
produces a finely focused beam of electrons called the primary electron beam.
These electrons pass through electromagnetic lenses and are directed over the
surface of the specimen.
The primary electron beam knocks electrons out of the surface of the
specimen, and the secondary electrons thus produced are transmitted to an
electron collector, amplified, and used to produce an image on a viewing screen
or photographic plate.
The image is called a scanning electron micrograph.
This microscope is especially useful in studying the surface structures of intact
cells and viruses.
 In practice, it can resolve objects as close together as 10 nm, and objects are
generally magnified 1000 to 10,000 X
 Scanning electron Microscopy
Specimen preparation for SEM is relatively easy, and in some cases, air-dried
material can be examined directly.
However, microorganisms usually must first be fixed, dehydrated, and dried to
preserve surface structure and prevent collapse of the cells when they are
exposed to the SEM's high vacuum.
Before viewing, dried samples are mounted and coated with a thin layer of
metal
to prevent the buildup of an electrical charge on the surface and to give a better
image
To create an image, the SEM scans a narrow, tapered electron beam back and
forth over the specimen .
When the beam strikes a particular area, surface atoms discharge a tiny
shower of electrons called secondary electrons, and these are trapped by a
detector.
Secondary electrons entering the detector strike a scintillator, causing it to emit
light flashes that a photomultiplier converts to an electrical current and amplifies.
The signal is sent to a cathode-ray tube and produces an image that can
be viewed or photographed.
The number of secondary electrons reaching the detector depends on the
nature of the specimen's surface.
 Scanning electron Microscopy
When the electron beam
strikes a raised area, a large
number of secondary
electrons enter the detector;
in contrast, fewer electrons
escape a depression in the
surface and reach the
detector.
Thus raised areas appear
lighter on the screen and
depressions are darker. A
realistic three-dimensional
image of the
microorganism's surface
results.
The actual in situ location
of microorganisms in
ecological niches such as the
human skin and the lining of
the gut also can be examined
 Atomic Force Microscope (AFM)
The atomic force microscope (AFM) is a type of scanning probe microscope
whose primary roles include measuring properties such as magnetism, height,
friction. 

Scanning probe microscopy (SPM) is a branch of microscopy that forms

images of surfaces using a physical probe that scans the specimen.

The resolution is measured in a nanometer, which is much more accurate and

effective than the optical diffraction limit.

It uses a probe for measuring and collection of data involves touching the
surface that has the probe.

An image is formed when the scanning probe microscope raster-scans the probe
over a section of the sample, measuring its local properties concurrently.

A raster scan, or raster scanning, is the rectangular pattern of image capture and
reconstruction in television (Displaying or capturing a video image line by line).
 Atomic Force Microscope (AFM)
 They also have piezoelectric elements, which are electric charges that
accumulate in selected solid materials like DNA, biological proteins, crystal, etc,
to enable tiny accurate and precise movement during scanning upon an electric
command. 

Piezoelectric elements have a unique characteristic whereby the element

elongates or vibrates when an external voltage is applied, similar to how it
generates electricity when external pressure is applied

The Atomic Force Microscope was invented in 1982, by scientists working in

IBM, just after the invention of the Scanning tunneling Microscope in 1980 by
Gerd Binnig and Heinrich Rohler by IBM Research in Zurich. That is when
Binnig later invented the Atomic Force Microscope, and it was first used
experimentally in 1986. It was put on the market for commercial sale in 1989.
 Atomic Force Microscope (AFM)

PZT, or lead zirconate titanate (Pb[Zr(x)Ti(1-x)]O3), is one of the world's most

widely used piezoelectric ceramic materials.
 Atomic Force Microscope (AFM)
The Atomic Force Microscope works on the principle measuring intermolecular
forces and sees atoms by using probed surfaces of the specimen in nanoscale. Its
functioning is enabled by three of its major working principles that include
Surface sensing,
Detection, and
Imaging.

The Atomic Force Microscope (AFM) performs surface sensing by using a

cantilever (an element that is made of a rigid block like a beam or plate, that
attaches to the end of support, from which it protrudes making a perpendicularly
flat connection that is vertical like a wall).

The cantilever has a sharp tip that scans over the sample surface, by forming an
attractive force between the surface and the tip when it draws closer to the sample
surface. When it draws very close making contact with the surface of the sample,
a repulsive force gradually takes control making the cantilever avert from the
surface.
 Atomic Force Microscope (AFM)
During the deflection of the cantilever away from the sample surface, there is a
change in direction of reflection of the beam, and a laser beam detects the
aversion, by reflecting off a beam from the flat surface of the cantilever.

 Using a positive-sensitive photo-diode (PSPD- a component that is based on

silicon PIN diode technology and is used to measure the position of the integral
focus of an incoming light signal), it tracks these changes of deflection and
change in direction of the reflected beam and records them.

The Atomic Force Microscope (AFM) takes the image of the surface
topography of the sample by force by scanning the cantilever over a section of
interest.

Depending on how raised or how low the surface of the sample is, it determines
the deflection of the beam, which is monitored by the PSDP.

The microscope has a feedback loop that controls the length of the cantilever
tip just above the sample surface, therefore, it will maintain the laser position thus
generating an accurate imaging map of the surface of the image.
 Atomic Force Microscope (AFM)

Applications of Atomic Force Microscope

This type of microscopy has been used in various disciplines in natural science
such as solid-state physics, semiconductor studies, molecular engineering,
polymer chemistry, surface chemistry, molecular biology, cell biology, medicine,
and physics.

Some of these applications include:

Identifying atoms from samples

Evaluating force interactions between atoms
Studying the physical changing properties of atoms
Studying the structural and mechanical properties of protein complexes and
assembly, such as microtubules.
used to differentiate cancer cells and normal cells.
Evaluating and differentiating neighboring cells and their shape and cell wall
rigidity.
A summary of various types of Microscopes
A summary of various types of Microscopes
A summary of various types of Microscopes
Clinically important viruses
Hepatitis
 Hepatitis
Hepatitis is a liver inflammation, commonly caused by an infectious agent.
Inflammation  is part of the complex biological response of body tissues to
harmful stimuli, such as pathogens, damaged cells, or irritants, and is a
protective response involving immune cells, blood vessels, and molecular
mediator
Hepatitis sometimes results in acute illness followed by destruction of
functional liver anatomy and cells, a condition known as cirrhosis.
Hepatitis due to infection can cause chronic or acute disease, and some forms
lead to liver cancer.
Acute illnesses generally develop suddenly and last a short time, often only a
few days or weeks. Chronic conditions develop slowly and may worsen over an
extended period of time—months to years
Although many viruses and a few bacteria can cause hepatitis, restricted group
of viruses is often associated with liver disease.
Hepatitis viruses A, B, C, D, and E are phylogenetically diverse viruses but
share in common their ability to infect the liver.
Hepatitis A and E viruses, although occasionally transmitted person to person,
are more commonly transmitted by food (hepatitis A virus) or water (hepatitis E
virus).
Fulminant  is a medical descriptor for any event or process that occurs suddenly and escalates
quickly, and is intense and severe to the point of lethality, i.e., it has an explosive character.
Hepatitis
 Hepatitis
Fibrosis is the formation of an abnormally large amount of scar tissue in the liver.
It occurs when the liver attempts to repair and replace damaged cells. Many
conditions can damage the liver. Fibrosis itself causes no symptoms, but
severe scarring can result in cirrhosis, which can cause symptoms.
 Viral Hepatitis
• When they occur, the signs and symptoms of viral
hepatitis can include:
– Fever
– Fatigue
– Loss of appetite
– Nausea
– Vomiting
– Abdominal pain
– Jaundice
– Dark urine
– Clay-colored stool
– Joint pain
Hepatitis A

VPg (viral protein genome-linked) 

 Hepatitis A-Genome
HAV (Hepatitis A Virus) is a distinct member of the picornavirus family, assigned
to genus hepatovirus.
HAV is a 27 nm to 32 nm spherical particle with icosahedral symmetry containing
a linear single-stranded RNA genome with a size of 7.5 kb and non-enveloped
 HAV genome can be divided into three parts a 5′ noncoding region (NCR) that
comprises approximately 10% of the genome, is uncapped, and is covalently linked
at the 5′ terminus to viral protein VPg a single open reading frame that appears to
encode all of the viral proteins, with regions designated as P1 for capsid proteins
and P2 and P3 for nonstructural proteins a short 3′ NCR terminating in a polyA tail.
 Hepatitis A-replication
The regions P1 contains four segments for structural proteins which make up
the capsid protein; 1A-VP4, 1B- VP2, 1C-VP3, 1D-VP1.
P2 comprises of three non structural proteins; 2A, 2B, 2C which play a role in
viral replication.
P3 makes up four non structural proteins
 3A- anchors the replication complex to cell membrane
 3B- it is VPg protein
 3C- it is cysteine protease that cleaves the protein from polypeptides
 3D- it is RNA dependent RNA Polymerase.
HAV is spread primarily through the ingestion of fecally contaminated food or
water.
Once HAV reaches the intestine, it is thought to be absorbed into the
bloodstream and to reach the liver through the portal system.
Attachment of the virus to host cell receptors (HAV cr-1) mediates endocytosis
of the virus into the host cell possibly by clathrin- dependent endocytosis.
 Hepatitis A-replication
Upon endosomal acidification, the capsid undergoes a conformational change
and release VP4 that opens a pore in the host endosomal membrane and the viral
genomic RNA penetrates into the host cell cytoplasm.
VPg protein is removed from the viral RNA, which is translated into a
processed polyproten.
The IRES (Internal ribosome entry site) allows direct translation of the
polyprotein.
A ds RNA genome is synthesized from the genomic ssRNA(+).
The dsRNA genome is transcribed thereby providing viral mRNAs/new
ssRNA(+) genomes.
New genomic RNA is believed to be packaged into preassembled procapsids.
Cell lysis occurs and virus is released.
Viral replication occurs primarily within hepatocytes and the secretion of virus
into bile results in large quantities of virus being shed in the faeces.
HAV replication in the liver triggers a substantial immune response, both
humoral and cell mediated.
CD8 +, cytotoxic T cells that are capable of lysing autologous HAV infected
cells , but not of controlling uninfected cells, are present both in circulation and
in the liver at the site of disease.
 Hepatitis B
Here our focus is on hepatitis viruses transmitted by direct contact, with
the major focus on hepatitis B, the causative agent of “bloodborne hepatitis.”
The incidence of hepatitis A and B, the most common forms, has decreased
significantly in the past 20 years because of effective vaccines and increases
in surveillance. And, by comparison to hepatitis A and B, hepatitis C
infections have risen significantly in recent years
Infection with hepatitis B virus (HBV) is called bloodborne hepatitis (or
serum hepatitis) because it is transmitted in blood or in body fluids in contact
with blood.
HBV is a hepadnavirus, a partially double-stranded DNA virus.

Hepadnaviruses have small, enveloped, spherical virions (virus particles)

that are about 40–48 nm in diameter. The capsid (the protein shell
surrounding the viral nucleic acids) contains a circular double-stranded
DNA molecule with a single-stranded DNA region and a DNA-dependent
DNA polymerase
The mature virus particle containing the viral genome is called a Dane particle
-a spherical particle found in the serum in hepatitis B that is the virion of the
causative double-stranded DNA virus.
The outer capsule contains the antigen called the hepatitis B surface antigen
(HBs-Antigen). This is also called as Australia antigen (HBsAg).
The inner core contains HBV core antigen one is called HBc Ag and another
antigen is incorporated into the Core antigen is called HBeAg.
The HBcAg is a very small amount so not detectable but its Antibody is found
in circulation.
HBV causes acute, often severe disease that can lead to liver failure and death.
Chronic HBV infection can lead to cirrhosis and liver cancer
HBV is transmitted by a parenteral route, which means “outside the gut.”
The main means of HBV transmission is from blood transfusions, contact with
infected blood in a hypodermic needle, and from mother to child during
childbirth.
HBV may also be transmitted through exchanges of body fluids during sex.
 The number of new HBV infections has remained low and more or less
constant since the year 2000.
Nevertheless, over 100,000 people worldwide die yearly from liver failure or
liver cancer caused by chronic HBV infection
Hepatitis B
 HBV antigens
Hepatitis B Surface Antigen (HBsAg)
• HBsAg appears first in the blood, so this is a more common test.
• HBsAg rises before the appearance of clinical signs and symptoms
appear and is detectable.
• The peak is during the first week of symptoms.
• It returns to a normal level by the time jaundice subsides.
• If it persists then the patient will be a carrier or may develop chronic
hepatitis.

Hepatitis B Surface Antibody (HBsAb)

• This antibody appears after roughly 4 weeks, after the disappearance
of HBsAg.
• It indicates the end of the acute phase and the patient complete
recovery from the infection.
• The patient will develop immunity to HBV infection.
• After vaccination, there is the appearance of HBsAb
 HBV antigens
Hepatitis Core Antigen (HBcAg)
• This is not detectable because of the very small quantity and it is
incorporated with HBeAg.
Hepatitis B Core Antibody (HBcAb )
• This antibody appears after one month of infection.
• In acute infection, this will be HBcAb-IgM type and later replaced by
HBcAb-IgG type.
• This antibody persists in circulation for several years.
• This antibody will be present in chronic hepatitis cases.
• In the window period when HBsAg is negative and still there are no
HBsAb, then this is the antibody present in the patient.
• HbcAb-IgM is the marker in the window period in acute infections.
• This is also called as the window period marker.
Hepatitis Be Antigen (HBeAg)
• HBeAg is a marker of the infectivity.
• HBeAg appears immediately after the appearance of HBsAg.
• HBeAg indicates early and acute disease.
• HBeAg positive in chronic hepatitis patients is a sign of a bad prognosis.
• The persistence of HBeAg indicates the development of chronic hepatitis.
Hepatitis B E Antibody (HBeAb)
•The appearance of HBeAb is a sign of recovery.
•This antibody shows the end of the acute phase
Hepatitis C
Hepatitis C virus (HCV) is also transmitted parenterally. HCV generally
produces a mild or even asymptomatic disease at first, but later on up to 85% of
those infected develop chronic hepatitis, with up to 20% proceeding to chronic
liver disease and cirrhosis.
Chronic infection with HCV leads to hepatocarcinoma (liver cancer) in 3–5%
of infected individuals.
The latency period for development of cancer can be several decades after the
primary infection.
Large numbers of HCV-related deaths occur annually as a result of chronic HCV
infections that develop into liver cancer.
This was formally called non-A, non-B viral infection because no test was
available. This was suspected by the exclusion of HBV and HAV.
Hepatitis C virus (HCV) is a hepatotropic virus.
• HCV  is a small enveloped virus measuring 55 to 65 nm.
• HCV is a single-stranded RNA virus of the Flaviviridae family.
• This was formally called non-A, non-B viral infection because no test was
available. This was suspected by the exclusion of HBV and HAV.

Hepatotropic -Having an especial attraction or affinity for, or an effect on, the liver.

 Replication of Hepatitis C Virus
The HCV lifecycle begins with the attachment of a virion to its specific receptors
on hepatocytes: the high-density lipoprotein receptor , scavenger receptor class B
type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the
known cellular receptors initiating the attachment step of HCV infection.
Viral internalisation is through endocytosis of the bound virion in a clathrin-
coated endosome, and fusion of viral and endosomal membranes and that the
nucleocapsid is released into the cytoplasm.
The virus is then uncoating to free its genomic RNA, and the HCV genomic
RNA is used both for polyprotein translation and replication in the cytoplasm.
Replication of HCV takes place in membranous webs associated with the NS4B
protein and cellular endoplasmic reticulum (ER).
HCV replication is catalyzed by the NS5B protein.
NS4B initiates the formation of replication complex that supports HCV
replication.
The viral RNA-dependent RNA polymerase, first copies the positive sense RNA
into a negative-sense RNA intermediate and then copies the negative-sense RNA
into more positive sense RNA.
 Replication of Hepatitis C Virus
The core protein, however, remains within the cytoplasm after cleavage from E1,
E1 and E2 are embedded in the ER membrane, and their extracellular domains are
glycosylated.
Nascent genomes can then be translated, further replicated or packaged within
new virus particles.
New virus particles are thought to bud into the secretory pathway and are
released at the cell surface.
Hepatitis D virus (HDV) is a defective virus that lacks genes encoding its own
capsid. HDV is also transmitted by parenteral routes, but because it is a defective
virus, it cannot replicate and form an intact virion unless the cell is also infected
with HBV.
A virion is an entire virus particle consisting of an outer protein shell called a
capsid and an inner core of nucleic acid (either ribonucleic or deoxyribonucleic
acid—RNA or DNA). The core confers infectivity, and the capsid provides
specificity to the virus
The HDV genome replicates independently but relies on HBV to produce capsid
proteins (which are the same as those used by HBV) to form infectious virions.
Thus, HDV infections are always coinfections with HBV.
The HDV (hepatitis delta virus) is a small, spherical virus with a 36 nm diameter.
It has an viral envelope containing host phospholipids and three kinds of HBV
envelope protein – large, medium, and small hepatitis B surface antigens; this
surrounds an inner ribonucleoprotein (RNP) particle.
The ribonucleoprotein particle contains the genome surrounded by about 200
molecules of hepatitis D antigen (HDAg) for each genome.
The central region of HDAg has been shown to bind RNA. Several interactions
are also mediated by a coiled-coil region at the N terminus of HDAg.


 Hepatitis D virus (HDV)
The HDV genome is negative sense, single-stranded, closed circular RNA; with
a genome of approximately 1700 nucleotides, HDV is the smallest "virus" known
to infect animals.
It has been proposed that HDV may have originated from a class of plant
pathogens called viroids, which are much smaller than viruses.
 Its genome is unique among animal viruses because of its high GC nucleotide
content.
Its nucleotide sequence is about 70% self-complementary, allowing the genome
to form a partially double-stranded, rod-like RNA structure
Hepatitis
 D virus (HDV) structure. (A)
Schematic representation of HDV viral
particle. HDV virion contains an envelope
derived from the endoplasmic reticulum, in
which are embedded the three forms (S, M and
L) of hepatitis B virus (HBV) envelope protein,
HBs antigen (HBsAg). HDV genome is a
circular single stranded RNA of negative
polarity associated to the two forms of delta
antigen (L-HDAg and S-HDAg) forming a
ribonucleoproteic complex.
 Schematic representation of the delta virion and its replication cycle

(1) The virion attaches to the hepatocyte via an interaction between large-HBsAg and an
uncharacterised membrane receptor in the host cell;
(2) the virion enters the cell and is uncoated;
(3) the RNP is targeted to the nucleus;
(4) genomic RNA is transcribed in the nucleus to form antigenomic RNA, which forms the
template for replication of new transcripts of the circular genome, and mRNA, which contains
the open reading frame;
(5) the mRNA is exported to the cytoplasm where it is translated at the
endoplasmic reticulum to form new molecules of hepatitis D antigen; \
(6) the new antigen molecules return to the nucleus where the small-
HDAg isoform supports further genome replication, and where both
forms of hepatitis D antigen associate with new transcripts of genomic
RNA to form new RNPs;
(7) RNPs are exported to the cytoplasm where large-HDAg facilitates
association with HBV envelope proteins in the ER to form new virus
particles;
(8) these particles bud through an intermediate compartment;
(9) they are then exported from the hepatocyte via the trans-Golgi
network to re-infect further cells.

RNP=ribonucleoprotein. mRNA=messenger RNA. HBV=hepatitis B

virus. HBsAg=hepatitis B surface antigen. HDAg=hepatitis D antigen.
ER=endoplasmic reticulum
 Structure of Hepatitis E Virus

Schematic illustration of non-enveloped and quasi-enveloped HEV particles as well

as enveloped virus. The putative model of quasi-enveloped HEV virion includes
ORF3 product in its envelope as the existence of pORF3 has been confirmed by
capturing quasi-enveloped HEV virion with anti-pORF3 antibodies and further
supported by prediction of a putative transmembrane region in the N-terminal of
 Structure of Hepatitis E Virus

HEV is classified in the Calciviridae family because of its structural similarity to

other calciviruses; however, it is now the sole member of the Hepeviridae family.
The virus is non enveloped (naked) with icosahedral symmetry measuring 27-30 nm
diameter.
The genome is single stranded RNA genome with positive polarity and measure
about 7.2 kb in length.
Genomic RNA is polyadenylated and contains 3 ORFs.
ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and
ORF3 encodes a small multifunctional protein.
The ORF2 and ORF3 proteins are translated from a single, bicistronic mRNA.
Located near the 5′-end, ORF1 encodes a non-structural polyprotein with multiple
functional domains, including those for methyltransferase, protease, helicase, and
polymerase.
The viral capsid protein (CP) is encoded by ORF2 near the 3′-end.
ORF3, which partially overlaps with the other 2 ORFs, codes for an immunogenic
protein of unknown function.
 Structure of Hepatitis E Virus

ORF3 encodes a 113 or 114 aa phosphoprotein, depending upon the genotype.

The ORF2 capsid protein, HEV-CP, contains a total of 660 amino acid residues.
At the HEV-CP N terminus is a signal peptide followed by an arginine-rich domain
that potentially play a role in viral RNA encapsidation during assembly.
HEV-CP is a key antigen that stimulates the host immune response, and 6 antigenic
domains have been identified.
One neutralization site has been mapped to the polypeptide region between amino
acids 452 and 617.
The mechanisms underlying HEV replication are poorly understood.
The HEV capsid protein is believed to bind to a cellular receptor to initiate viral
entry and replication.
ORF2 peptide-binding experiments suggested that the C-terminal region of ORF2
may mediate virus entry by binding to heat shock cognate protein 70 (HSC70) on the
cell surface.
Additionally, HSPGs have been identified as attachment receptors that are located
on the cell surface.
After virus entry into permissive cells, the HEV genomic RNA is uncoated by
unknown mechanisms.
 Structure of Hepatitis E Virus
After uncoating, virion releases the positive-sense genomic RNA into the
cytoplasm of the cell.
The positive-sense genomic viral RNA serves as the template to translate the
ORF1 nonstructural polyprotein in the cytoplasm.
The viral RdRp synthesizes an intermediate, replicative negative-sense RNA
from the positive-sense genomic RNA that serves as the template for the
production of positive-sense, progeny viral genomes.
The ORF2 and ORF3 proteins are translated from the subgenomic, positive-
stranded RNA, and the ORF2 capsid protein packages the genomic viral RNA and
assembles new virions.
The nascent virions are transported to the cell membrane.
The ORF3 protein facilitates the trafficking of the virion, and the nascent virions
are released from the infected cells by lysis.
The pathogenesis of hepatitis E is poorly understood.
HEV infection can lead to more severe, acute liver disease in pregnant women
or patients with underlying chronic liver diseases and sometimes progress to
fulminant hepatic failure.
 Other Aspects of Hepatitis Syndromes
Hepatitis is an acute disease of the liver, a vital organ that plays a role in
several key metabolic processes, including carbohydrate, lipid, and protein
syntheses, as well as detoxification and many other functions.
Symptoms of hepatitis include fever, jaundice (yellowing of the skin and the
whites of the eyes) and liver enlargement and cirrhosis.
All hepatitis viruses cause similar acute symptoms and cannot be readily
distinguished based on clinical findings alone.
Chronic hepatitis infections, usually caused by HBV or HCV, are often
asymptomatic or produce very mild symptoms, but nonetheless cause serious
liver disease, even in the absence of liver cancer
Diagnosis of hepatitis is based on a combination of clinical symptoms and
laboratory tests that assess liver function, especially key liver enzymes.
Cirrhosis is diagnosed by visual examination of biopsied liver tissue.
Virus-specific molecular assays are typically used to confirm a diagnosis,
positively identify the infectious agent, and determine a course of treatment;
isolation and culture of hepatitis viruses is usually not attempted
 Other Aspects of Hepatitis Syndromes
Many of the immunological and molecular diagnostic tools are used in
hepatitis diagnoses.
These include enzyme immunoassays that target viral-specific proteins
or antiviral antibodies in a blood sample, immunoblots (Western
blots), and immunofluorescence (microscopic) methods.
Polymerase chain reaction (PCR) tests are also used to detect hepatitis viral
genomes in blood or in liver tissue obtained by biopsy.
Infection with HAV or HBV can be prevented with effective vaccines.
No effective vaccines are available for the other hepatitis viruses. For those
unvaccinated, the practice of universal precautions will prevent infection.
The precautions prescribe a high level of vigilance and aseptic handling and
containment procedures to deal with patients, body fluids, and infected waste
materials .
Most treatment of hepatitis is supportive, providing rest and time for the
immune system to attack the infection and allow liver damage to be
repaired. In some cases, in particular for HBV infections, some antiviral drugs
are available that offer effective treatment.
Clinically important viruses
Influenza
 Difference Between Cold or FLU

A cold is a milder respiratory illness than the flu. While cold symptoms can make
you feel bad for a few days. Flu symptoms can make you feel quite ill for a few
days to weeks.

Cold symptoms usually begin with a sore throat, which usually goes away after a
day or two. Nasal symptoms, runny nose, and congestion follow, along with a
cough by the fourth and fifth days. Fever is uncommon in adults, but a slight fever
is possible. Children are more likely to have a fever with a cold.
With cold symptoms, your nose teems with watery nasal secretions for the first
few days. Later, these become thicker and darker.

Whether a person has typical seasonal flu or swine flu, the symptoms seem to be
quite similar. Flu symptoms are usually more severe than cold symptoms and
come on quickly. Symptoms of swine flu and seasonal flu include sore throat,
fever, headache, muscle aches and soreness, congestion, and cough. Swine flu in
particular is also associated with vomiting and diarrhea.
 Influenza
Influenza, commonly known as "the flu", is an infectious disease caused by
an influenza virus. 
Symptoms can range from mild to severe and commonly include:
high fever, runny nose, sore throat, muscle and joint pain, headache, coughing,
and feeling tired.
 These symptoms typically begin two days after exposure to the virus and
most last less than a week, although the coughing may last for more than two
weeks.
In children, there may be diarrhea and vomiting, but these are not common in
adults.
 Diarrhea and vomiting occur more commonly in gastroenteritis, which is an
unrelated disease sometimes referred to as "stomach flu" or the "24-hour flu“.
Complications of influenza may include viral pneumonia, secondary bacterial
pneumonia, sinus infections, and worsening of previous health problems such
as asthma or heart failure
Three of the four types of influenza viruses affect humans: Type A, Type B,
and Type C. Type D has not been known to infect humans, but is believed to
have the potential to do so.
 Influenza
Usually, the virus is spread through the air from coughs or sneezes. This is
believed to occur mostly over relatively short distances.
It can also be spread by touching surfaces contaminated by the virus and then
touching the eyes, nose, or mouth.
 A person may be infectious to others both before and during the time they are
showing symptoms.
The infection may be confirmed by testing the throat, sputum, or nose for the
virus.
 A number of rapid tests are available; however, people may still have the
infection even if the results are negative. A type of polymerase chain reaction that
detects the virus's RNA is more accurate.
Frequent hand washing reduces the risk of viral spread, as does wearing
a surgical mask.
 Yearly vaccinations against influenza are recommended by the World Health
Organization (WHO) for those at high risk, and by the Centers for Disease
Control and Prevention (CDC) for those six months of age and older.
 The vaccine is typically effective against three or four types of influenza and is
usually well tolerated. 
 Antiviral medications such as the neuraminidase inhibitor oseltamivir, among
others, have been used to treat influenza
 Influenza
One of the most important Emerging and Reemerging infectious diseases
Causes high morbidity and mortality in communities (epidemic) and worldwide
(pandemic)
Epidemics are associated with excess mortality
Caused by a virus belonging to the MYXOVIRUS group which comprises of
Orthomyxovirus and Paramyxovirus
Influenza virus is an Orthomyxovirus
First isolated from a pig in 1931 (swine flu)
Isolated from human in 1933
The virus is spread from person- to- person through respiratory secretions either
as droplets (close contact) or as airborne infection by droplet nuclei suspended in
the air. Incubation period 1-3 days
The clinical picture of influenza is nonspecific.
Influenza-like illness can be caused by many microbial agents other than
influenzavirus, such as adenovirus, parainfluenza viruses, coronavirus,
Mycoplasma pneumoniae, Chlamydia pneumoniae, beta-hemolytic streptococcus.
Since the clinical picture of influenza is nonspecific, its specific diagnosis must
be confirmed by laboratory tests.
This is usually made by virus isolation, identification of specific antigens or
antibody rise.
Influenza
Influenza A virus

•The type A viruses are the most virulent human pathogens among the three
influenza types and cause the most severe disease. Type A flu or influenza A
viruses are capable of infecting people as well as animals.
•Wild aquatic birds are the natural hosts for a large variety of influenza A.
•The influenza A virus can be subdivided into different serotypes based on the
antibody response to these viruses. The serotypes that have been confirmed in
humans, ordered by the number of known human pandemic deaths, are:
•H1N1, which caused Spanish flu in 1918, and the 2009 flu pandemic
•H2N2, which caused Asian Flu in 1957
•H3N2, which caused Hong Kong Flu in 1968
•H5N1, a current pandemic threat
•H7N7, which has unusual zoonotic potential
•H1N2, endemic in humans and pigs
•H9N2
•H7N2
•H7N3
•H10N7
Strains of influenza are characterized by two proteins that are on the
outer surface of the virus: hemagglutinin and neuraminidase. This is
where the "H" and "N" in the name come from. The proteins can be
seen in the picture below, represented by the blue "spikes" on the
outside of the virus. Both of these proteins are required for the virus
to cause an infection and perform complementary functions. The
hemagglutinin is critical for the virus to be able to attach to, and
then enter the cell. Without hemagglutinin, the entire process of
infection could not be initiated. 
Influenza B virus

Influenza B almost exclusively infects humans and is less common than

influenza A. This type of influenza mutates at a rate 2–3 times lower than type
A. This reduced rate of antigenic change, combined with its limited host range
ensures that pandemics of influenza B do not occur.

Influenza virus C

Influenza C virus, which infects humans, dogs and pigs, sometimes causing both
severe illness and local epidemics. However, influenza C is less common than
the other types and usually only causes mild disease in children.
Structure

•Influenza viruses A, B and C are very similar in overall structure. The virus
particle is 80–120 nanometres in diameter and usually roughly spherical, although
filamentous forms can occur. These filamentous forms are more common in
influenza C, which can form cordlike structures up to 500 micrometres long on the
surfaces of infected cells.
The viral particles of all influenza Viruses are similar
in composition. These are made of a viral envelope
containing two main types of glycoproteins, wrapped
around a central core.
The central core contains the viral RNA genome and
other viral proteins that package and protect this RNA
RNA tends to be single stranded but in special cases i
is double. Unusually for a virus, its genome is not a
single piece of nucleic acid; instead, it contains seven
or eight pieces of segmented negative-sense RNA,
each piece of RNA contains either one or two genes.
For example, the influenza A genome contains 11
genes on eight pieces of RNA, encoding for 11
proteins: hemagglutinin (HA), neuraminidase (NA),
nucleoprotein (NP), M1, M2, NS1, NS2(NEP), PA,
PB1, PB1-F2 and PB2
Structure

Hemagglutinin & Neuraminidase

Hemagglutinin (HA) and neuraminidase (NA) are the two large

glycoproteins on the outside of the viral particles. HA is a lectin
that mediates binding of the virus to target cells and entry of
the viral genome into the target cell, while NA is involved in the
release of progeny virus from infected cells, by cleaving sugars
that bind the mature viral particles.

Thus, these proteins are targets for antiviral drugs.

Furthermore, they are antigens to which antibodies can be
raised.
 Antigenic Variation
Influenza viruses tend to undergo changes from time to time. There are two types
of changes: (1) antigenic shift, (2) antigenic drift. These changes in the antigenic
characteristics of influenza viruses determine the extent and severity of influenza
epidemics
Antigenic Shift
1. This term denotes MAJOR changes in hemagglutinin and neuraminidase
resulting from reassortment of gene segments involving two different
influenza viruses.
2. When this occurs, worldwide epidemics may be the consequence since the
entire population is susceptible to the virus.
3. Hemagglutinins are glycoproteins which cause red blood cells (RBCs) to
agglutinate or clump together (note that agglutination is one of three steps in
the more complex process of coagulation)
4. Influenza hemagglutinin (HA) is a homotrimeric glycoprotein found on the
surface of influenza viruses and is integral to its infectivity.
5. Neuraminidase (Sialidase) enzymes are glycoside hydrolase enzymes that
cleave (cut) the glycosidic linkages of neuraminic
acids. Neuraminidase enzymes are a large family, found in a range of
organisms.
 Antigenic Variation

Reassortment is the mixing of the genetic material of a species into new

combinations in different individuals. Several different processes contribute
to reassortment, including assortment of chromosomes, and chromosomal
crossover.
Mutation is an important source of RNA virus diversity that is made possible by
the error-prone nature of RNA synthesis. Viruses with segmented genomes, such
as influenza virus, have another mechanism for generating diversity: reassortment.
When an influenza virus infects a cell, the individual RNA segments enter the
nucleus. There they are copied many times to form RNA genomes for new
infectious virions. The new RNA segments are exported to the cytoplasm, and
then are incorporated into new virus particles which bud from the cell.
If a cell is infected with two different influenza viruses, the RNAs of both
viruses are copied in the nucleus. When new virus particles are assembled at the
plasma membrane, each of the 8 RNA segments may originate from either
infecting virus. The progeny that inherit RNAs from both parents are called
reassortants.,
 Antigenic Variation
The below shows a cell that is co-infected with two influenza viruses L and M. The infected
cell produces both parental viruses as well as a reassortant R3 which inherits one RNA segment
from strain L and the remainder from strain M.
 Antigenic Drift
• This term denotes MINOR changes in
hemagglutinin and neuraminidase of
influenza virus.
• This results from mutation in the RNA
segments coding for either the HA or NA
• This involves no change in serotype;
there is merely an alteration in amino
acid sequence of HA or NA leading to
change in antigenicity.
Reservoirs of Influenza Viruses

• Aquatic birds
• Pigs
• Humans
 Types of Vaccine

• Inactivated, consisting of (1) whole-

virus, (2) subvirion, (3) purified
surface antigen. Only subvirion or
purified antigen should be used in
children. Any of the three can be used
for adults.
• Live attenuated
Influenza Vaccine, who should
receive it
• Persons 65 yrs or
older
• Persons with heart,
pulmonary, renal and
metabolic diseases.
• Persons in nursing
homes and other long-
term care facilities
• Persons 6 mos-18 yrs
old receiving aspirin
Influenza vaccine recipients--
continued
• Women in 2nd or 3rd
trimester of pregnancy
during flu season.
• Household members
of persons in high-
risk groups
• Health care workers
and others providing
essential community
services.
 Antiviral Drugs

• Amantadine, rimantadine. Effective for

prevention and treatment of flu A only.
• Zanamivir, oseltamivir are approved for
treatment of uncomplicated flu A & B;
oseltamivir also approved for prophylaxis.
• Prophylaxis must be continued throughout
the epidemic; treatment must begin within
24 hrs of onset of illness.
 HIV
• What is HIV?
• How is HIV transmitted?
• Basic characteristics of HIV disease course.
• Reasons for the rapid rate of HIV evolution.
• Where did HIV come from?
• When did HIV first jump into humans?

http://pathmicro.med.sc.edu/lecture/hiv9.htm
 What is HIV?

To understand what HIV and AIDS are, let’s break it down:

Causative agent:

H – Human – This particular virus can only infect human beings.

I – Immunodeficiency – HIV weakens your immune system by

destroying important cells that fight disease and infection.
A "deficient" immune system can't protect you.
V – Virus – A virus can only reproduce itself by taking over a cell in
the body of its host.
 Disease?
AIDS

A – Acquired – AIDS is not something you inherit from your parents

like eye color.
You acquire AIDS.

I – Immuno – Your body's immune system includes all the organs and
cells that work to fight off infection or disease.
D – Deficiency – You get AIDS when your immune system is
"deficient,“ or isn't working the way it should.
S – Syndrome – A syndrome is a collection of symptoms and signs of
disease. AIDS is a syndrome, rather than a single disease.

It is a complex illness with a wide range of symptoms.

What is HIV?
Human immunodeficiency virus or HIV is a type of virus
(from the Latin “virus” referring to poison).

Viruses are:

Small
-Generally too small to see with a regular light microscope (20- 400 nm diameter)
If a cell was a football stadium then a small virus would be around the size of a
football.
-Can only replicate in living cells
-Some can survive for long periods of time outside cells, but cannot replicate that way.
-Made up of Nucleic acids (DNA/RNA) and proteins
-different from protein-only “prions” or nucleic acid-only “viroids”.
-Thousands of very different types of virus exist and HIV is a particular type termed a
“retrovirus”.
What is HIV?
-HIV is a retrovirus in the genus Lentivirus.
-Many retroviruses have the potential to cause cancer and produce dire (serious
condition), often fatal diseases and are capable of altering the host’s DNA in profound
ways.
-They are named “retroviruses” because they reverse the usual order of transcription.
-They contain an unusual enzyme called reverse transcriptase (RT) that catalyzes the
replication of double-stranded DNA from single-stranded RNA.
-The association of retroviruses with their hosts can be so intimate that viral genes
are permanently integrated into the host genome.
-It has become increasingly evident that retroviral sequences are integral parts of host
chromosomes.
-Not only can this retroviral DNA be incorporated into the host genome as a provirus
that can be passed on to progeny cells, but also some retroviruses transform cells and
regulate certain host genes.

A provirus is a virus genome that is integrated into the DNA of a host cell. In
the case of bacterial viruses, proviruses are often referred to as prophages.
However, it is important to note that proviruses are distinctly different from
prophages and these terms should not be used interchangeably
What is HIV?
-There are two major types of HIV—namely HIV-1, which is the
dominant form in most of the world, and HIV-2.
- Genetic sequencing of HIV-1 shows that it is most related to simian
immunodeficiency viruses in chimpanzees, while HIV-2 evolved from
related viruses in sooty mangabeys, a type of monkey found in Africa.
-Both highlight the evidence that HIV in humans was derived from a
zoonotic primate virus.
-HIV and other retroviruses display structural features typical of
enveloped RNA viruses
-The outermost component is a lipid envelope with transmembrane
glycoprotein spikes and knobs that mediate viral adsorption to the host
cell. HIV can only infect host cells that present the required receptors,
which is a combination receptor consisting of the CD4 marker plus a
coreceptor called CCR-5.
-The virus uses these receptors to gain entrance to several types of
leukocytes and tissue cells
 The general structure of HIV

(a) The envelope contains two types of glycoprotein (GP) spikes, two identical RNA
strands, and several molecules of reverse transcriptase, protease, and integrase
encased in a protein capsid. (b) The snug attachment of HIV glycoprotein molecules
(GP-41 and GP-120) to their specific receptors on a human cell membrane. These
receptors are CD4 and a coreceptor called CCR-5 (fusin) that permit docking with
the host cell and fusion with the cell membrane
Where did HIV come from?
Zoonosis = Cross-species transmission event

http://what-when-how.com/medical-microbiology-and-infection/zoonoses-systemic-infection/
• Origin HIV: most researchers feel the virus originated
in west Africa, somewhere
• between 40 and 100 years ago. This infection was
contained in a small area, probably
• remote, until the 1950’s and 1960’s. Both social and
political upheaval in Africa as well
• as the development of rapid and wide spread travel
contributed to its spread. (this is
• theoretical – an emerging disease – contained for long
time and then spread)
Origin of aids; controversial, similar to
SIV
Where did HIV come from?
HIV entered the human population from primates, which harbor a related virus known as SIV
(simian immunodeficiency virus). This probably occurred during the butchering and
consumption of monkey meat in Africa.

HIV-1 group M,
HIV “family tree” which is the most
prevalent HIV strain,
jumped from
chimpanzees into
humans.

http://www.avert.org/hiv-types.htm HIV-2 originated in sooty

mangabeys and is
responsible for fewer
infections than HIV-1

New viruses are still being transferred from primates to humans, and have the potential to
cause new diseases and epidemics.
 Origins of human AIDS viruses.

Sharp P M , and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841

Origins of human AIDS viruses. Old World monkeys are naturally infected with more than 40
different lentiviruses, termed simian immunodeficiency viruses (SIVs) with a suffix to denote their
primate species of origin (e.g., SIVsmm from sooty mangabeys). Several of these SIVs have
crossed the species barrier to great apes and humans, generating new pathogens (see text for
details). Known examples of cross-species transmissions, as well as the resulting viruses, are
highlighted in red.
Origin and Spread of HIV
Pathogenesis and Virulence Factors

HIV enters a mucous membrane or the skin and travels to dendritic cells, a type
of phagocyte living beneath the epithelium.
In the dendritic cell, the virus grows and is shed from the cell without killing it.
The virus is amplified by macrophages in the skin, lymphoid organs, bone
marrow, and blood.
One of the great ironies of HIV is that it infects and destroys many of the very
cells needed to combat it, including the helper (T4 or CD4) class of lymphocytes,
monocytes, macrophages, and even B lymphocytes.
The virus is adapted to docking onto its host cell’s surface receptors . It then
induces viral fusion with the cell membrane and creates syncytia (multinucleated
giant-cell formation )
Once the virus is inside the cell, its reverse transcriptase converts its RNA into
DNA. Although initially it can produce a lytic infection, in many cells, it enters a
latent period in the nucleus of the host cell and integrates its DNA into host DNA
This latency accounts for the lengthy course of the disease. Despite being
described as a “latent” stage, research suggests that new viruses are constantly
being produced and new T cells are constantly being manufactured, in an
ongoing race that ultimately the host cells lose (in the absence of treatment).
Pathogenesis and Virulence Factors

The primary effects of HIV infection causes harm to T cells and the central
nervous system.
The death of T cells and other white blood cells results in extreme leukopenia
and loss of essential T4 memory clones and stem cells.
Leukopenia is a condition where a person has a reduced number of white blood
cells.
The viruses also cause formation of giant T cells and other syncytia, which
allow the spread of viruses directly from cell to cell, followed by mass
destruction of the syncytia.
The destruction of T4 lymphocytes paves the way for invasion by opportunistic
agents and malignant cells.
The central nervous system is affected when infected macrophages cross the
bloodbrain barrier and release viruses, which then invade nervous tissue.
Studies have indicated that some of the viral envelope proteins can have a
direct toxic effect on the brain’s glial cells and other cells.
The secondary effects of HIV infection are the opportunistic infections and
malignancies that occur as the immune system becomes progressively crippled by
viral attack.
The general multiplication cycle of HIV
The general multiplication cycle of HIV
 Reasons for the rapid rate of HIV evolution

• HIV mutates every time it replicates

• HIV replicates in billions of cells simultaneously every

day

• HIV therefore evolves around 1 MILLION TIMES

• faster than mammalian genes

http://preprod.www.tibotec.com/content/backgrounders/www.tibotec.com/hiv_lifecycle.html
 Untreated HIV infection is a constant battle between the virus and the host immune system,
with BILLIONS of new infected cells and virus particles produced and cleared EVERY DAY
in each infected person.

http://www.healthhype.com/cd4-count-dropping-viral-load-stable-in-hiv-infection-graph.html
http://www.niaid.nih.gov/topics/HIVAIDS/Understanding/Biology/pages/clinicalcourse.aspx
How is HIV transmitted?
http://aids.gov
 Transmission

In general, HIV is spread only by direct and rather specific routes.
Because the blood of HIV-infected individuals harbors high levels of
free virus in both very early and very late stages of infection and high
levels of infected leukocytes throughout infection, any form of intimate
contact involving transfer of blood (trauma, needle sharing) can be a
potential source of infection.
Semen and vaginal secretions also harbor free virus and infected white
blood cells; thus, they are significant factors in sexual transmission.
The virus can be isolated from urine, tears, sweat, and saliva but in
such small numbers that these fluids are not considered sources of
infection.
Because breast milk contains significant numbers of leukocytes,
neonates who escaped infection prior to and during birth can still
become infected through nursing
 Culture and Diagnosis
A person is diagnosed as having HIV infection if he or she has tested positive for
exposure to the human immunodeficiency virus. This diagnosis is not the same as
having AIDS.
In 2012, the U.S. Preventive Services Task Force recommended that all people
between the ages of 15 and 64 be tested for HIV.
People outside of that age group who are at high risk, as well as pregnant
women, should also be tested.
Current testing guidelines call for plasma or serum samples being analyzed for
antibodies to HIV as well as for HIV p24 antigen.
 If the test is negative, there are no further tests performed. If it is positive,
further tests are run to determine if the virus is HIV-1 or HIV-2.
In 2012, the FDA approved an over-the-counter testing method called
OraQuick.
It is available at drugstores and uses a mouth swab to detect antibodies to the
virus in 20 to 40 minutes. There is some controversy over the easy accessibility to
the test (without counseling) because users may not understand that their—or their
partners’—results may not be accurate if they are inside the period before
antibodies develop.
However, public health officials believe that wider access to testing will help
decrease the spread of the virus by those who do not know they have it.
 Culture and Diagnosis
In the United States, people are diagnosed with AIDS if they
meet the following criteria:
(1) They are positive for the virus and
(2) they fulfill one of these additional criteria:
• They have a CD4 (helper T cell) count of fewer than
200 cells per microliter of blood.
• Their CD4 cells account for fewer than 14% of all
lymphocytes.
• They experience one or more of a CDC-provided list
of AIDS-defining illnesses (ADIs).
 Treatment
Clear-cut guidelines exist for treating people who test HIVpositive. These guidelines are
updated regularly.
The newer recommendations call for treatment to begin immediately after HIVdiagnosis.
In addition to antiviral chemotherapy, AIDS patients
should receive a wide array of drugs to prevent or treat a variety of
opportunistic infections and other ADIs such as wasting disease.
These treatment regimens vary according to each patient’s profile
and needs
Mechanisms of action of anti-HIV
drugs
Mechanisms of action of anti-HIV
drugs