UNIT-1 VIROLOGY

Shapes & sizes & components of genomes in virus-

Components of Genome- Viral genomes consist of DNA or RNA only, never

both. DNA and RNA molecules can be double stranded or single stranded, linear or circular (Fig.
1.6), segmented (composed of multiple pieces of nucleic acid) or nonsegmented. The meaning of
the term genomic segment could be confusing as its use is driven by both nomenclature
principles and historic reasons. Strictly speaking, a genome segment is an individual and unique
piece of nucleic acid among multiple pieces comprising one whole viral genome. For example,
the influenza A virus has segmented genome comprised of eight ssRNA segments (Fig. 1.5).
Herpesviruses, which have nonsegmented genomes composed of one linear dsDNA molecule,
have the so-called UL (unique long) and US (unique short) segments, corresponding to
regions/portions of their genomes flanked by repeats. The HIV genome can be somewhat
confusing too with each virion carrying two copies of the same ssRNA molecule. The HIV
genome is considered nonsegmented, and the two ssRNA molecules are called copies not
segments. In many viruses the genome ends contain repeated sequences, chemical modifications,
or secondary structures, which often have regulatory functions. Genomes are tightly packed
inside the capsids and frequently the genome and the capsid are collectively called nucleocapsid.
Amazingly, viruses are able to execute productive infection and of course make us sick with very
limited genetic information. The flu virus genome, for example, contains only 15,000
nucleotides. For comparison the human genome is 3,200,000,000 nucleotides or approximately
200,000 times longer. Needless to say, viruses have to be superefficient, in their quest to invade
the host cell and to propagate. Bacteriophage Qβ is among the smallest RNA viruses with a
genome built from 4217 nucleotides and only 4 genes. Among the smallest known animal DNA
viruses is TT virus whose genome is comprised of less than 4000 nucleotides and 4 predicted
genes. On the opposite side of the scale is the giant Megavirus chilensis with genome as large as
1.3 MB coding for 1000 genes. What functions viral genomes code for is a tantalizing question.
Analysis of viral genome sequences revealed that approximately 80% of the viral genomes code
for virus-specific genes, many of which have no known homologues or known function.
Shapes

Figu
re 10.2.110.2.1A: Sizes and Shapes of Viruses (Animal RNA Viruses)
 Figure 10.2.110.2.1B: Sizes and
Shapes of Viruses (Animal DNA Viruses)

Figure 10.2.110.2.1C: Sizes and Shapes of Viruses (Bacteriophages)

a. Helical viruses consist of nucleic acid surrounded by a hollow protein cylinder or

capsid and possessing a helical structure (Figure 10.2.210.2.2A).
 Figure 10.2.210.2.2:Viral Structure
(Helical Virus). (B) Viral Structure (Polyhedral Virus), (C) Viral Structure (Enveloped Helical
Virus), D: Viral Structure (Enveloped Polyhedral Virus), (F) Viral Structure (Binal) Illustration
of a T-even bacteriophage consisting of a head, sheath, and tail.

Chemical composistion-RNA Virus Genomes

RNA viruses, comprising 70% of all viruses, vary remarkably in genome structure . Because of
the error rate of the enzymes involved in RNA replication, these viruses usually show much
higher mutation rates than do the DNA viruses. Mutation rates of 10-4 lead to the continuous
generation of virus variants which show great adaptability to new hosts. The viral RNA may be
single-stranded (ss) or double-stranded (ds), and the genome may occupy a single RNA segment
or be distributed on two or more separate segments (segmented genomes). In addition, the RNA
strand of a single-stranded genome may be either a sense strand (plus strand), which can function
as messenger RNA (mRNA), or an antisense strand (minus strand), which is complementary to
the sense strand and cannot function as mRNA protein translation . Sense viral RNA alone can
replicate if injected into cells, since it can function as mRNA and initiate translation of virus-
encoded proteins. Antisense RNA, on the other hand, has no translational function and cannot
per se produce viral components.
DsRNA viruses, e.g., members of the reovirus family, contain 10, 11 or 12 separate genome
segments coding for 3 enzymes involved in RNA replication, 3 major capsid proteins and a
number of smaller structural proteins. Each segment consists of a complementary sense and
antisense strand that is hydrogen bonded into a linear ds molecule. The replication of these
viruses is complex; only the sense RNA strands are released from the infecting virion to initiate
replication.
The retrovirus genome comprises two identical, plus-sense ssRNA molecules, each monomer 7–
11 kb in size, that are noncovalently linked over a short terminal region. Retroviruses contain 2
envelope proteins encoded by the env-gene, 4–6 nonglycosylated core proteins and 3 non-
structural functional proteins (reverse transcriptase, integrase, protease: RT, IN, PR) specified by
the gag-gene . The RT transcribes the viral ssRNA into double-stranded, circular proviral DNA.
This DNA, mediated by the viral integrase, becomes covalently bonded into the DNA of the host
cell to make possible the subsequent transcription of the sense strands that eventually give rise to
retrovirus progeny. After assembly and budding, retroviruses show structural and functional
maturation. In immature virions the structural proteins of the core are present as a large precursor
protein shell. After proteolytic processing by the viral protease the proteins of the mature virion
are rearranged and form the dense isometric or cone-shaped core typical of the mature virion,
and the particle becomes infectious

. DNA Virus Genomes

Most DNA viruses contain a single genome of linear dsDNA. The papovaviruses, comprising
the polyoma- and papillomaviruses, however, have circular DNA genomes, about 5.1 and 7.8 kb
pairs in size. DsDNA serves as a template both for mRNA and for self-transcription. Three or 2
structural proteins make up the papovavirus capsid: in addition, 5-6 nonstructural proteins are
encoded that are functional in virus transcription, DNA replication and cell transformation.
Single-stranded linear DNA, 4–6 kb in size, is found with the members of the Parvovirus family
that comprises the parvo-, the erythro- and the dependoviruses. The virion contains 2–4
structural protein species which are differently derived from the same gene product . The adeno-
associated virus (AAV, a dependovirus) is incapable of producing progeny virions except in the
presence of helper viruses (adenovirus or herpesvirus). It is therefore said to be replication
defective.
Circular single-stranded DNA of only 1.7 to 2.3 kb is found in members of the Circovirus family
which comprise the smallest autonomously propagated viruses. The isometric capsid measures
17 nm and is composed of 2 protein species only
Genome size

Genome size varies greatly between species. The smallest—the ssDNA circoviruses,
family Circoviridae—code for only two proteins and have a genome size of only two kilobases;
[75]
the largest—the pandoraviruses—have genome sizes of around two megabases which code
for about 2500 proteins.[66] Virus genes rarely have introns and often are arranged in the genome
so that they overlap.[76]
In general, RNA viruses have smaller genome sizes than DNA viruses because of a higher error-
rate when replicating, and have a maximum upper size limit.[24] Beyond this, errors when
replicating render the virus useless or uncompetitive. To compensate, RNA viruses often have
segmented genomes—the genome is split into smaller molecules—thus reducing the chance that
an error in a single-component genome will incapacitate the entire genome. In contrast, DNA
viruses generally have larger genomes because of the high fidelity of their replication enzymes.
[77]
Single-strand DNA viruses are an exception to this rule, as mutation rates for these genomes
can approach the extreme of the ssRNA virus case.[78]
Genetic mutation and recombination

Antigenic shift, or reassortment, can result in novel and highly pathogenic strains of human flu
Viruses undergo genetic change by several mechanisms. These include a process called antigenic
drift where individual bases in the DNA or RNA mutate to other bases. Most of these point
mutations are "silent"—they do not change the protein that the gene encodes—but others can
confer evolutionary advantages such as resistance to antiviral drugs.[79][80] Antigenic shift occurs
when there is a major change in the genome of the virus. This can be a result
of recombination or reassortment. When this happens with influenza viruses, pandemics might
result.[81] RNA viruses often exist as quasispecies or swarms of viruses of the same species but
with slightly different genome nucleoside sequences. Such quasispecies are a prime target for
natural selection.[82]
Segmented genomes confer evolutionary advantages; different strains of a virus with a
segmented genome can shuffle and combine genes and produce progeny viruses (or offspring)
that have unique characteristics. This is called reassortment or 'viral sex'.[83]
Genetic recombination is a process by which a strand of DNA (or RNA) is broken and then
joined to the end of a different DNA (or RNA) molecule. This can occur when viruses infect
cells simultaneously and studies of viral evolution have shown that recombination has been
rampant in the species studied.[84] Recombination is common to both RNA and DNA viruses.[85]
[86]

Coronaviruses have a single-strand positive-sense RNA genome. Replication of the genome is

catalyzed by an RNA-dependent RNA polymerase. The mechanism of recombination used by
coronaviruses likely involves template switching by the polymerase during genome replication.
[87]
This process appears to be an adaptation for coping with genome damage.[88]
Replication cycle

A typical virus replication cycle

Some bacteriophages inject their genomes into bacterial cells (not to scale)
Viral populations do not grow through cell division, because they are acellular. Instead, they use
the machinery and metabolism of a host cell to produce multiple copies of themselves, and they
assemble in the cell.[89] When infected, the host cell is forced to rapidly produce thousands of
copies of the original virus.[90]
Their life cycle differs greatly between species, but there are six basic stages in their life cycle: [91]
Attachment is a specific binding between viral capsid proteins and specific receptors on the host
cellular surface. This specificity determines the host range and type of host cell of a virus. For
example, HIV infects a limited range of human leucocytes. This is because its surface
protein, gp120, specifically interacts with the CD4 molecule—a chemokine receptor—which is
most commonly found on the surface of CD4+ T-Cells. This mechanism has evolved to favour
those viruses that infect only cells in which they are capable of replication. Attachment to the
receptor can induce the viral envelope protein to undergo changes that result in the fusion of viral
and cellular membranes, or changes of non-enveloped virus surface proteins that allow the virus
to enter.[92]
Penetration or viral entry follows attachment: Virions enter the host cell through receptor-
mediated endocytosis or membrane fusion. The infection of plant and fungal cells is different
from that of animal cells. Plants have a rigid cell wall made of cellulose, and fungi one of chitin,
so most viruses can get inside these cells only after trauma to the cell wall.[93] Nearly all plant
viruses (such as tobacco mosaic virus) can also move directly from cell to cell, in the form of
single-stranded nucleoprotein complexes, through pores called plasmodesmata.[94] Bacteria, like
plants, have strong cell walls that a virus must breach to infect the cell. Given that bacterial cell
walls are much thinner than plant cell walls due to their much smaller size, some viruses have
evolved mechanisms that inject their genome into the bacterial cell across the cell wall, while the
viral capsid remains outside.[95]
Uncoating is a process in which the viral capsid is removed: This may be by degradation by viral
enzymes or host enzymes or by simple dissociation; the end-result is the releasing of the viral
genomic nucleic acid.[96]
Replication of viruses involves primarily multiplication of the genome. Replication involves the
synthesis of viral messenger RNA (mRNA) from "early" genes (with exceptions for positive-
sense RNA viruses), viral protein synthesis, possible assembly of viral proteins, then viral
genome replication mediated by early or regulatory protein expression. This may be followed,
for complex viruses with larger genomes, by one or more further rounds of mRNA synthesis:
"late" gene expression is, in general, of structural or virion proteins.[97]
Assembly – Following the structure-mediated self-assembly of the virus particles, some
modification of the proteins often occurs. In viruses such as HIV, this modification (sometimes
called maturation) occurs after the virus has been released from the host cell. [98]
Release – Viruses can be released from the host cell by lysis, a process that kills the cell by
bursting its membrane and cell wall if present: this is a feature of many bacterial and some
animal viruses. Some viruses undergo a lysogenic cycle where the viral genome is incorporated
by genetic recombination into a specific place in the host's chromosome. The viral genome is
then known as a "provirus" or, in the case of bacteriophages a "prophage".[99] Whenever the host
divides, the viral genome is also replicated. The viral genome is mostly silent within the host. At
some point, the provirus or prophage may give rise to the active virus, which may lyse the host
cells.[100] Enveloped viruses (e.g., HIV) typically are released from the host cell by budding.
During this process, the virus acquires its envelope, which is a modified piece of the host's
plasma or other, internal membrane.

Lytic & Lysogenic cycle- keypoints-

  latency: The ability of a pathogenic virus to lie dormant within a cell.
 bacteriophage: A virus that specifically infects bacteria.
 lytic cycle: The normal process of viral reproduction involving penetration of the cell
membrane, nucleic acid synthesis, and lysis of the host cell.
 lysogenic cycle: A form of viral reproduction involving the fusion of the nucleic acid of a
bacteriophage with that of a host, followed by proliferation of the resulting prophage.

Different Hosts and Their Viruses-

Viruses are often very specific as to which hosts and which cells within the host they will infect.
This feature of a virus makes it specific to one or a few species of life on earth. So many
different types of viruses exist that nearly every living organism has its own set of viruses that
try to infect its cells. Even the smallest and simplest of cells, prokaryotic bacteria, may be
attacked by specific types of viruses.
Bacteriophages are viruses that infect bacteria. Bacteriophages may have a lytic cycle or a
lysogenic cycle, and a few viruses are capable of carrying out both. When infection of a cell by a
bacteriophage results in the production of new virions, the infection is said to be productive.

Figure- Lytic versus lysogenic cycle: A temperate bacteriophage has both lytic and
lysogenic cycles. In the lytic cycle, the phage replicates and lyses the host cell. In the
lysogenic cycle, phage DNA is incorporated into the host genome, where it is passed on to
subsequent generations. Environmental stressors such as starvation or exposure to toxic
chemicals may cause the prophage to excise and enter the lytic cycle.

Lytic Cycle-With lytic phages, bacterial cells are broken open (lysed) and destroyed after
immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find
new hosts to infect. An example of a lytic bacteriophage is T4, which infects E. coli found in the
human intestinal tract. Lytic phages are more suitable for phage therapy.

Lysogenic Cycle-

In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages
able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with
host DNA and replicate along with it fairly harmlessly, or may even become established as a
plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of
nutrients; then, the endogenous phages (known as prophages) become active. At this point they
initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows
the host cell to continue to survive and reproduce, the virus is reproduced in all of the cell’s
offspring. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle
is the phage lambda of E. coli.

Latency Period-

Viruses that infect plant or animal cells may also undergo infections where they are not
producing virions for long periods. An example is the animal herpes viruses, including herpes
simplex viruses, which cause oral and genital herpes in humans. In a process called latency, these
viruses can exist in nervous tissue for long periods of time without producing new virions, only
to leave latency periodically and cause lesions in the skin where the virus replicates. Even though
there are similarities between lysogeny and latency, the term lysogenic cycle is usually reserved
to describe bacteriophages.

 Keypoints-The lytic cycle involves the reproduction of viruses using a host cell to
manufacture more viruses; the viruses then burst out of the cell.
 The lysogenic cycle involves the incorporation of the viral genome into the host cell
genome, infecting it from within.

Isolation and purification of viruses and components-

Virus Isolation - Viruses are obligate intracellular parasites that require living cells in order
to replicate. Generally cell culture, embryonated eggs and small laboratory animals are used for
the isolation of viruses. Embryonated eggs are very useful for the isolation of influenza and
paramyxoviruses. Although laboratory animals are useful in isolating different kind of viruses,
cell culture is still a preferred way for virus isolation in many of the laboratories. For primary
cell cultures, tissue fragments are first dissociated into small pieces with the help of scissors and
addition of trypsin. The cell suspension is then washed couple of times with minimal essential
media and seeded into a flat-bottomed glass or plastic container bottle after resuspending it with
a suitable liquid medium and fetal calf serum. The cells are kept in incubator at 370C for 24 to
48hrs depending on the cell type. This allows the cells to attach the surface of the container and
its division following the normal cell cycle. Cell cultures are generally of 3 types –
Primary culture – These are prepared directly from animal or human tissues and can be
subcultured only once or twice e.g. chicken embryo fibroblast.

2. Diploid cell culture – They are derived from neonatal tissues and can be subcultured 5-10
times. e.g. human diploid fibroblasts cells.

3. Continuous cells – They are derived from tumor tissues and can be subcultured more than
10 times. e.g. Vero, Hep2, Hela

Specimens containing virus should be transported to the laboratory as soon as possible upon
being taken. Oral or cloacal swabs should be collected in vials containing virus transport
medium. Body fluids and tissues should be collected in a sterile container and sealed properly.
If possible all the samples should be maintained and transported in a cold condition for higher
recovery rates. Upon receipt, the samples should be inoculated into cell culture depending on
the history and symptoms of the disease. The infected cell culture flask should be observed
every day for any presence of cytopathic effect (CPE). Certain kind of samples, such as faeces
and urine are toxic to the cell cultures and may produce a CPE-like effect. When virus specific
CPE is evident, it is advised to passage the infected culture fluid into a fresh cell culture. For
cell-associated viruses such as cytomegaloviruses, it is required to trypsinize and passage the
intact infected cells. Viruses such as adenovirus can be subcultured after couple of time freezing
and thawing of the infected cells.

Purification of virus and components:

Ultracentrifugation: The viruses are usually purified with the help of ultracentrifugation.
The machine is capable of rotating the samples at 20,000-100,000 rpm under the density
gradient of CsCl2 or sucrose. Density at which viruses neither sink nor float when suspended in
a density gradient is called as buoyant density. The rate at which viral particles sediment under
a defined gravitational force is called as sedimentation coefficient. The basic unit is the
Svedberg (S) which is 10-13 sec. The S value of a virus is used to estimate its molecular weight.

Types of sedimentation medium

A. Sucrose cushions or gradient -A fixed concentration or a linear gradient of sucrose

is used. Increasing the density and viscosity of the medium decreases the rate at which
virus sediments through them. In general a "cushion" of sucrose is prepared at the
bottom of the centrifuge tube and the sample containing virus is overlaid over the
cushion. Since most viruses have greater densities than sucrose, separation is based on
S values. This method can be used to separate molecules with relatively close S values.
Sometime glycerol is also used in place of sucrose.
 B. CsCl2 gradient centrifugation -A linear gradient of CsCl2 in buffer is prepared in the
ultracentrifuge tube. As the concentration of the CsCl2 is increased the density of the
medium increases in the tube so that density is low at the top and high at the bottom.
Viral particle centrifuged through this medium will form a band at a position equal to
their buoyant density. These are useful to separate viruses of different densities.
Limitation of this method is that CsCl2 can permanently inactivate some viruses.

Other techniques for separation: Viruses can also be separated by electrophoresis

and column chromatography but these are not the preferred way to separate virus while
sometimes they are used to separate viral nucleic acids or proteins. Both the methods separate
the virus on the basis of charge and/or size. Virus contains a variety of charged macromolecule
on its surface which contributes to its electrophoretic mobility or ion-exchange characteristics.
Viruses are sometimes ligated with the charged group to be separated by ion exchange
chromatography. Molecular sieve chromatography can also be used to purify the viruses where
large pores are formed with the help of special agarose through which virus particles can enter.

Purity of viruses: Many methods are used to assess the purity of virus. The ratio of UV
absorption at 260 and 280 nm during a spectrophotometric analysis (260/280) is a
characteristic feature to measure the purity of a virus sample and is dependent on the amount
of nucleic acid and protein present in the virion. Serological methods such as enzyme-linked
immunosorbent assay (ELISA), radioimmuno precipitation assay (RIPA), western blot, virus
neutralization test (VNT), and complement fixation are also used to check the puirity of a virus
sample. These methods require antibodies specific to viral proteins that may be monoclonal
(single type of antibody specific to a single viral protein) or polyclonal (several different
antibodies that may recognize several viral proteins or epitopes). Plaque assay is also
performed in order to isolate the single colony from a pool of quasispecies viruses..

UNIT-2
Introduction -Since viruses are intracellular parasites, they rely on their host cells for the
energy, macromolecular synthesis machinery and the work benches for genome replication and
particle assembly. Because of this dependence, viruses have evolved a myriad of mechanisms for
exploiting normal host cell functions. Often this exploitation is associated with damage to the
host cell which may be one of the major factors in the pathology and disease caused by viruses.
The material in this entry is confined to model systems of virus–host cell interactions that
involve the infection by animal viruses of cells in culture.

Types of Virus Infections- When a virus infects a cell, the outcomes that may
occur can be grouped into several general categories which are determined by the particular
virus involved, as well as by the type of cell and its functional state.

1) Productive infections-Productive infections result in the formation of progeny virus

and usually cause the destruction of the host cell.
2) Persistant Infections- In some cases the host cells are not all destroyed, leading to
persistent infections in which the surviving cells multiply and continue to produce
progeny viruses. When persistent infections occur in which the viral genome is present
but no infectious virus is produced, these infections are referred to as latent infections. In
such infections some level of viral gene expression is usually detectable although virions
are not produced.
3) Transforming Infections-When genetic information of the virus is integrated as DNA
into the host cell genome or is carried as episomal DNA, transforming infections may
take place. Such infections can cause an oncogenic alteration of the growth properties of
the cell
4) Abortive infections -This occur when viruses infect cells that are nonpermissive or
only partially permissive. In this instance, the virus is able to enter the cell but because
some step essential for viral replication is absent, the replication cycle does not go to
completion and no progeny are produced. Such abortive infections may or may not cause
cell death.

NOTE-A few examples follow which demonstrate that different outcomes of infection are
dependent on the particular virus and host cell involved, as well as on the state of the host cell.
For example, influenza A virus causes a productive, cytolytic infection of a line of canine kidney
cells (MDCK). However, when the same virus infects the L cell line of mouse fibroblasts an
abortive infection occurs because of a block at the level of virion RNA replication. The same
mouse L cell line supports a productive, cytolytic infection by vesicular stomatitis virus (VSV).
However, if the L cells are pretreated with interferon, the functional state of the cells is altered;
the VSV replication cycle is blocked at the level of protein synthesis and an abortive infection
results. When VSV infects insect cell lines derived from Aedes or Drosophila, productive
noncytolytic infections occur. Continuous passage of the insect cell lines reveals that they have
become persistently infected and continuously produce infective virus without any signs of
cytopathology. Adeno-associated virus (AAV), a parvovirus, is capable of a productive or a
latent infection depending upon whether or not the host cells are co-infected with a helper virus
such as adenovirus. In some host cells, AAV can cause a latent infection by integrating its DNA
into the host cell genome.
Effects of Virus Infection on the Host Cell-
Effects on host cell morphology and viability-The most readily recognized effects of
viruses on host cells are those that involve morphologic changes or cell death. Enders defined
viral cytopathogenicity as ‘the capacity to induce any demonstrable departure from the normal
either in the morphological or functional properties of cells’. The space available for this entry
precludes a comprehensive survey of all the cytopathic effects induced by infection with the
various families of animal viruses. However, one of the most striking observations that emerges
from an overview of the effects of viruses on host cells is how little is known of the mechanisms
by which viruses induce cytopathology. The production of cytopathic effects has been observed
with most families of viruses and in many cases the viral gene(s) involved or implicated in these
morphological changes has been defined. However, in most cases the mechanisms responsible
for cell destruction have not been identified. It is fair to say that one of the most fundamental
questions of virology, namely, how viruses kill cells, remains for the most part unanswered.

Effects on host cell macromolecular synthesis-Many of the investigations of the effect

of virus infection on the host cell have centered on virus-induced alterations of host cell
macromolecular synthesis. While these studies are important to an understanding of the viral
growth cycle and have yielded significant insights into the control of host cell gene expression,
there is no direct evidence that inhibition at this level is the direct cause of visible cytopathology
or cell death. In fact, treatment of host cells with drugs such as actinomycin D and
cycloheximide, which inhibit nucleic acid and protein synthesis, does not mimic the
morphological changes produced by virus infection. Nevertheless, viruses do employ a variety of
strategies to affect the host cell at the level of gene expression.

Effects on host cell DNA and RNA synthesis-A variety of DNA and RNA viruses are
capable of affecting gene expression by directly altering the host cell genome. For example,
the host cell DNA is degraded after infection with poxviruses. Herpes-, picorna- and
reoviruses cause a displacement of the cellular chromatin, while an inhibition of host DNA
synthesis has been reported following infection with herpes-, pox-, adeno-, picorna-, reo-,
alpha- and rhabdoviruses. This inhibition of host DNA synthesis may be a direct effect of a
virus factor in the nucleus or a secondary consequence of the inhibition of host cell protein
synthesis.

Effects on host cell protein synthesis-Although much effort has been directed at
understanding the effect of virus infection on host protein synthesis, it is unlikely that an
inhibition of host protein synthesis is required for successful virus replication. Many viruses,
such as paramyxo-, papova- and retroviruses do not normally inhibit host protein synthesis
during their replication. Furthermore, mutant viruses that are defective in their ability to shut
down host protein synthesis are not necessarily defective for virus growth. In fact, VSV mutants
selected during a persistent infection have a reduced ability to inhibit the host's translational
machinery; nevertheless, these mutants grow to higher titer during a lytic growth cycle than the
parental wild-type virus. With several virus families, infection causes a selective inhibition of the
translation of host cell mRNA. Such viruses include picorna-, pox-, herpes-, adeno-, rhabdo-,
reo- and orthomyxoviruses. In many cases this inhibition is accompanied by a decrease in the
overall rate of protein synthesis in the infected cell. It is likely that this overall inhibition occurs
at the level of initiation of protein synthesis since, where it has been examined, the average size
of the polysomes in the infected cells is reduced.

Viral Infection- Establishment of antiviral state-The action of interferons (IFNs)

on virus-infected cells and surrounding tissues elicits an antiviral state that is characterized by
the expression and antiviral activity of IFN-stimulated genes. In turn, viruses encode
mechanisms to counteract the host response and support efficient viral replication, thereby
minimizing the therapeutic antiviral power of IFNs.

Interferons (IFNs), although best known for their antiviral properties, are potent regulators of cell
growth3 and have immunomodulatory activity. Indeed, an emerging theme is that these
cytokines are important regulators of innate and adaptive immune responses. Furthermore,
studies now highlight the importance of cross-talk between cellular regulatory pathways that
control IFN signalling, apoptosis, inflammation and the stress response . There are two main
types of IFN, type I and type II. Type I or 'viral' IFNs include IFN-α, IFN-β, IFN-ω and IFN-τ;
type II IFN is IFN-γ. Most types of cell can produce IFN-α and IFN-β, which are the best-
characterized type I IFNs, whereas IFN-γ is produced only by certain cells of the immune
system, including natural killer (NK) cells, CD4+ T helper 1 (TH1) cells and CD8+ cytotoxic T
cells. There are 14 different IFN-α genes, but only one IFN-β and one IFN-γ gene. IFNs mediate
their effects through interactions with type-specific receptors, which are different and non-
redundant for the type I and type II IFNs. IFNα/β-receptor-knockout mice (as well as IFN-γ-
receptor knockouts) cannot mount effective antiviral responses. The IFN receptors do not have
enzymatic activity, but they set in motion a complex signalling pathway that ultimately results in
the transcription of hundreds of IFN-stimulated genes (ISGs) . It is now clear that although IFN
levels markedly increase in response to virus infection, the sequence of events, types of IFN that
are produced and ISGs that are targeted have an important effect on the outcome . Type I
interferons (IFNs) are a group of antiviral cytokines that are induced during viral infection by
viral-replication products, such as double-stranded (ds)RNA. IFNs exert their biological
functions by binding to specific cell-surface receptors. In turn, this triggers the intracellular IFN
signalling pathway — mainly the JAK–STAT pathway — which eventually induces the
expression of a large number of IFN-stimulated genes (ISGs). The ISGs, the workhorses of the
IFN response, set up an antiviral, antiproliferative and immunoregulatory state in the host cells.
However, most, if not all, viruses have evolved a broad spectrum of strategies to block and
interfere with the IFN pathway. Common viral strategies include

blocking of IFN induction/expression

b | intercepting receptor binding of IFNs through viral decoy IFN receptors
c | perturbation of the intracellular IFN signalling pathway and
d | directly downregulating the level of expression of ISGs. ADAR, RNA-specific adenosine
deaminase; IRF, IFN-regulatory factor; JAK, Janus kinase; Mx, myxovirus-resistance proteins;
OAS, oligoadenylate synthetase; PKR, protein kinase; STAT, signal transducer and activator of
transcription.
INTERFERON-INDUCED PROTEINS AND THEIR ANTIVIRAL
ACTIVITIES-

The replication of a wide range of different DNA and RNA animal viruses is inhibited by IFN,
both in cell culture and in animals . For many of the viruses that are sensitive to the antiviral
action of IFN in cell culture systems, the primary step of the virus multiplication cycle inhibited
typically is the synthesis of viral polypeptides . Exceptions do exist, however. Papovaviruses and
certain retroviruses often are inhibited in IFN-sensitive cell systems at early and late steps,
respectively, of their multiplication cycles , myxoviruses and rhabdoviruses may be inhibited in
certain cell types at or before primary transcription , and adenoviruses and poxviruses can be
comparatively resistant to the antiviral actions of IFN because of virus-encoded antagonists .
Among the IFN-induced proteins implicated in the antiviral actions of IFNs in virus-infected
cells are PKR, the 2′,5′-oligoadenylate synthetase (OAS) and RNase L, the RNA-specific
adenosine deaminase (ADAR), and the Mx protein GTPases. Double-stranded RNA plays a
central role in modulating protein phosphorylation, RNA degradation, and RNA editing
catalyzed by the IFN-inducible enzymes: the PKR kinase, the OAS synthetases, and the ADAR1
deaminase . dsRNA interestingly also serves as a template for gene silencing . IFN also induces a
form of nitric oxide synthase (iNOS2) and the MHC class I and II molecules, all of which play
important roles in immune responses to infections.

Virus Infections: Escape, Resistance, and Counterattack-

Many viruses establish life-long infections in their natural host with few if any clinical
manifestations. The relationship between virus and host is a dynamic process in which the virus
has evolved the means to coexist by reducing its visibility, while the host immune system
attempts to suppress and eliminate infection without damage to itself. This short review describes
a variety of strategies that are employed by viruses to evade host immune responses. These
include virus-associated escape from T cell recognition, and resistance to apoptosis and
counterattack

Escape-It is well established that control of virus infection by the immune response normally
requires virus-specific cytotoxic T cells (CTL). CTL are primed by dendritic cells and other
professional antigen presenting cells which process viral proteins that are either endogenously
produced or phagocytosed from apoptotic infected cells (crosspriming). Following clonal
expansion, the primed CTL can act against virus by killing infected cells via perforin- and/or
Fas-dependent pathways before new virus particles are made. The killing takes less than 4 hr
following CTL recognition of viral peptides presented by MHC class I molecules at the surface
of the infected cells. In parallel with this direct form of attack, CTL also release cytokines and
chemokines that have antiviral activity To avoid CTL attack, viruses have developed many
tricks to ensure that the infected cells are not targeted by CTL. These include interference with
the peptide-presenting pathway and viral epitope mutation.
For example, herpes simplex virus (HSV) establishes life-long infection with resistance to CTL
recognition by inhibition of peptide transport from the cytosol into the endoplasmic reticulum
(ER) where MHC class I/peptide complexes are assembled.
Viral epitope mutation represents another way for a virus escape from CTL and is probably of
central importance in HIV infection. Because HIV reverse transcriptase is error prone, the high
virus turnover continually generates many possible mutations. Under the pressure of CTL
responses, viruses that contain mutated critical amino acids in epitopes recognized by CTL are
selected, provided the mutation is not harmful to the virus. Many studies have shown that
mutational escape of HIV-1 or SIV occurs during the course of infection in patients or an animal
model .

Resistance- Prolonging the survival of infected cells is clearly in the interest of infecting viruses,
as it will allow full maturation and dissemination. On the other hand, apoptosis of the infected
cells triggered by either immune attack or as a direct outcome of viral infection is an important
component of the host antiviral response. Therefore, it is not surprising that many viruses encode
viral proteins that inhibit this process . There are two main pathways for induction of apoptotic
cell death. One involves death receptors on the cell surface (TNF receptor family), TNF-R1, Fas,
DR4, and DR5 by interaction with their respective ligands TNF-α, Fas ligand (FasL), and
TRAIL. The second pathway involves the participation of mitochondria and is regulated by
members of Bcl-2 family including the antiapoptotic molecules, bcl-2 or bcl-XL, or by the
proapoptotic effectors, bad, bax, or bak. Both death receptor and mitochondria pathways share a
common set of caspases that cleave specific cellular substrates, leading to DNA fragmentation,
the hallmark of apoptosis. Viruses can block one or both pathways.
 Figure- Evasion of Immune Responses by Viruses in Human

Counterattack- There are therefore many examples of viruses that avoid CTL responses
and resist apoptosis, prolonging the life of the infected cells in the host. In some cases,
despite strong CTL responses in acute infection, the virus can lead to generalized
immunosuppression, for instance HIV-1, measles, or certain strains of lymphocytic
choriomeningitis virus (LCMV). In the case of HIV, this leads to an impaired CTL
response at the late stage of disease. This is associated with, though not necessarily
 caused by, widespread apoptosis in T cells . As the majority of apoptotic cells are not
HIV infected this apoptosis could include HIV-specific CTL. In this issue of Immunity,
Mueller et al. have compared the susceptibility of HIV- and CMV-specific CTL to Fas-
mediated apoptosis. Interestingly, they found that HIV-specific CTL were more prone to
apoptosis induced by anti-Fas antibody than CMV-specific CTL from the same patient .
This was further confirmed by coculture of HIV-specific CTL with macrophages infected
with HIV-1, which induces surface expression of FasL . Why are HIV- but not CMV-
specific CTL, which express comparable levels of Fas on the cell surface, more sensitive
to Fas-mediated killing? Mueller et al. showed HIV-specific CTL were mainly
CD45RA−CD62L−CD8+ T cells, whereas most CMV-specific CTL had a terminally
differentiated phenotype (CD45RA+CD62L−CD8+). Together with studies of , these data
suggest that HIV skews maturation of HIV-specific CTL, making them more vulnerable
to Fas-mediated apoptosis. HIV nef induces FasL expression on infected cells, and these
can therefore counterattack by killing the CTL through the Fas-FasL interaction
Furthermore, crosslinking of CD4 by HIV envelope protein (gp120) in the presence of
the HIV tat protein may also induce FasL expression and apoptosis of uninfected
CD4+ T cells . Additionally, interaction of HIV envelope protein with the chemokine
receptor CXCR4 on macrophages can trigger the death of CD8+ T cells by TNF-TNF-R
interaction . Thus, HIV encodes three different viral proteins which can respond to T cell
response by fighting

RT-PCR-
 Polymerase chain reaction (PCR) is a temperature-dependent nucleic acid
amplification technique used to amplify the DNA or RNA in vitro enzymatically.
 Reverse transcriptase polymerase chain reaction, RT-PCR, is a type of PCR
technique that enzymatically amplifies the RNA in vitro.

Objectives of RT-PCR-

1. To amplify the specific segment of RNA, resulting in billions of copies of a single RNA
segment.
2. To diagnose certain infections, genes, and study gene expression.

Principle of RT-PCR-

RT-PCR combines the reverse transcription process with the conventional PCR process. The
sample RNA is first converted to double-stranded DNA (complementary DNA) by reverse
transcriptase enzyme in the reverse transcription process. The cDNA can then be thermally
broken down into two single-stranded DNA templates. In these ssDNA templates, primers can
anneal to their complementary sequences based on the nucleic acid hybridization principle. DNA
polymerase then elongates the primer by sequentially adding the nucleotides to the 3’ end and
generates a dsDNA following the principle of DNA replication. These three processes,
 denaturation, annealing, and elongation, are repeated in a cyclic manner regulating the reaction
temperature and resulting in millions of copies of the cDNA.

Requirements (Enzymes) of RT-PCR-

1. Nucleic Acid Sample (Sample RNA)

RNA is the sample for RT-PCR, unlike other PCR techniques using DNA as their sample.
Mostly mRNA is used as the sample. The RNA will be converted into cDNA before
amplification.
2. Reverse Transcriptase Enzyme
It is an enzyme that catalyzes the formation of complementary DNA (cDNA) strands from the
RNA strand. It is also called RNA-dependent DNA polymerase enzyme and is responsible for
central-dogma reverse. It is the major component of RT-PCR as it converts sample RNA into
cDNA for amplification.
3. DNA Polymerase Enzyme
DNA polymerases are enzymes that catalyze the synthesis of complementary DNA strands by
assembling the nucleotides sequentially according to the template strand. Taq DNA polymerase,
the DNA polymerase enzyme extracted from the bacterium Thermus aquaticus, is the most
widely DNA polymerase as it is thermally stable and continues its activity after the repeated
cycle of heating and cooling.
4. Primers (Oligo (dT) primers, random primers, and sequence-specific
primers)
Three different types of primers are used in RT-PCR;
1. Random Primers
These are the short single-stranded sequences of 6 to 8 nucleotides that bind at the
complementary site of RNAs with or without poly(A) for cDNA synthesis using reverse
transcriptase.
2. Oligo (dT) Primers
They are oligonucleotides, mostly of 12 – 18 nucleotides, containing a segment of repeating
deoxythymidine (dT) which binds at the polyA tail of mRNA.
3. Sequence-specific Primers
These are the short single-stranded sequences of nucleotides that bind to the specific region of
interest of the sample RNA. It is mostly used in one-step RT-PCR.
5. Deoxynucleotide Triphosphates
Deoxynucleotide triphosphates (dNTPs) are artificially synthesized nucleotides that act as
building blocks for synthesizing cDNA and new cDNA strands during amplification. 4 different
dNTPs are used; deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP),
deoxythymidine triphosphate (dTTP), and Deoxycytidine triphosphate (dCTP).
 6. PCR Buffers and Other Chemicals
7. Thermocycler (PCR Machine)

Types of RT-PCR-

Based on whether the reverse transcription and the amplification steps occur either in a single
reaction (or tube) or in two separate reactions (or tubes), RT- PCR can be classified into two
types:
1. One-Step RT-PCR
It is a type of RT – PCR where the reverse transcription and the amplification reactions occur in
a single tube. All the required components are added in a single tube. First, reverse transcription
occurs, forming cDNA, which is then amplified in a PCR process.
Advantages of One–Step RT – PCR over Two–Step RT – PCR
1. It has a simple and easy handling setup.
2. It has higher accuracy and specificity.
3. It has a lesser chance of contamination.
4. It is a cheaper and faster method.
Disadvantages of One-Step RT-PCR over Two-Step RT-PCR
1. It detects fewer templates per reaction mixture due to using multiple chemicals in a single
reaction tube.
2. Due to lower template detection, it requires a larger template for starting.
3. It does not permit the storage and further analysis of the cDNA formed during the reaction.
4. There is a higher chance of primer – dimer and non–specific binding.
5. The chance of reaction failure is comparatively high
.

2. Two-Step RT-PCR-
It is another type of RT – PCR where the reverse transcription and the amplification process
occur in two separate tubes. In the first tube, a reverse transcription reaction takes place, yielding
cDNA. These cDNAs are then transferred to another tube where the PCR mixture is added, and
the cDNAs are amplified.
Advantages of Two-Step RT – PCR over One-Step RT – PCR
1. It allows us to store cDNA formed by reverse transcription.
2. It has higher efficiency, accuracy, and reliability and detects larger templates per reaction
mixture.
3. Comparatively, lower chance of reaction failure, non-specific binding, and primer–dimer
bonding.
Disadvantages of Two–Step RT – PCR over One-Step RT – PCR
1. There is a higher chance of contamination.
2. It is a more complex and tedious process requiring more resources and a well-trained person.
 Figure- One-step vs. Two-step RT-PCR

Steps/Procedure of RT-PCR-

The core procedure can be broadly classified into two phases; reverse transcription and
amplification. The procedure also varies on one-step and two-step RT – PCR. But, the general
steps involved in both of the types are the same and can be summarized into four stages;
Preparatory stage, reverse transcription, amplification, and product analysis stage, viz.:
1. Preparatory Stage
It is the initial stage where RNA extraction is done, and all the reaction mixture is prepared.
First, all materials are arranged, safety measures are taken, the PCR reaction preparation area is
cleaned, all the reagents are brought to working temperature, and the sample is extracted or
brought from storage.
In the one-step RT-PCR, sample RNA, reverse transcriptase enzyme, RNase H, primers, DNA
polymerase, dNTPs, buffers, and all other components are added in a specified and pre-
calculated amount in a single reaction tube. The tube is then loaded into a thermocycler for
further processing.
In the two-step, RT-PCR, sample RNA, reverse transcriptase, RNase H, primers, dNTPs, and other
buffers and chemicals for reverse transcription are loaded in a tube. Then the tube is subjected to a
specified temperature in a thermocycler where cDNAs are formed.
 2. Reverse Transcription
It is the primary step where the RNA is converted into cDNA, which then undergoes
amplification.
All the reaction mixture, including reverse transcriptase, RNase H, dNTPs mixture, primers,
nuclease-free water, reverse transcription buffer, and other components in one-step RT-PCR and
DNA polymerase and other amplification components in the two-step RT-PCR are added in a
tube and subjected to a temperature of 40 – 50°C for 10 minutes to 30 minutes in a thermocycler.
At this temperature, the primer will bind to the respective site of the RNA sample, and the
reverse transcriptase enzyme will synthesize cDNA by adding the free dNTPs.
3. Amplification
This step is similar to the amplification process of other PCR techniques for DNA amplification.
In a one-step RT-PCR, the same reaction mixture is subjected to an amplification process. At the
same time, in the two-step RT-PCR, the cDNA is isolated and placed in another tube where
DNA polymerase, primers, PCR buffer, dNTPs, and other chemicals are added. Then the tube is
placed in a thermocycler for amplification.
The amplification step includes denaturation, annealing, and elongation occurring cyclically one
after another for a certain number of cycles pre-programmed by the user.
4. Product Analysis Stage
It is the final step where the reaction mixture subjected to PCR is analyzed to confirm that
desired amplification is achieved. The gel electrophoresis method is mostly used for product
analysis. In real-time RT-PCR, there is no need for this additional step.

Applications of RT-PCR-

1. Study Gene Expression-

The Traditional Northern Blot technique requires a larger mRNA sample to analyze and study
the gene expression. However, using RT-PCR, we can amplify the minute mRNA sample and
study the sequence of nucleotides, thus analyzing the gene expression. It is used in studying and
identifying multidrug-resistant genes and their expressions in pathogens.
2. Identification of Unknown Species-
RT-PCR is used to identify viruses like HIV, SARS viruses, dengue viruses, HCV, etc. Besides,
other microorganisms and even higher organisms are identified by studying their rRNA and
mRNA.
3. Infectious Disease Diagnosis-
Diagnosis of different types of viral infection, bacterial infection, fungal and parasite infection,
cancer cell, and genetic diseases are done using the RT-PCR technique in clinical laboratories.
4. Gene Insertion and Gene Therapy Study-
RT-PCR is used to prepare cDNA from eukaryotic mRNA, which lacks introns and can be
inserted into prokaryotes. RT-PCR is used in monitoring the result of gene insertion and gene
therapy. These procedures are supposed to show particular gene expression and code for a
 particular protein, hence translating specific types of mRNA sequence. This specific mRNA
sequence can be analyzed using RT-PCR.
5. Study Mutation and Cancer Cells-
RT-PCR can detect and quantify tissue-specific mutant alleles. It can also detect any undesired
changes in the mRNA sequence and unique mRNAs, which are produced only by the different
types of cancer cells in our body.
6. Tools of Genetic Engineering and Viral Study-

Rapid Antigen Testing -

Rapid antigen testing is a diagnostic test that is used to detect the presence of a specific viral
or bacterial antigen in a sample, typically from the nasopharynx or oropharynx. The test is
called "rapid" because it can produce results within a short period of time, usually
15-30
minutes. Rapid antigen testing uses antibodies that are designed to bind specifically to the
target antigen, producing a visible signal such as a color change or line on a test strip.

Rapid antigen testing is commonly used for the diagnosis of infectious diseases, such as
influenza, strep throat, and COVID-19. These tests are generally less expensive and easier to
administer than other diagnostic tests, such as PCR tests. However, a negative result on a
rapid antigen test may need to be confirmed with a more sensitive test, especially if the
patient has symptoms or has been in close contact with someone with the disease.
Working of RAT
1. Sample Collection: The healthcare provider collects a sample from the patient using a
swab, typically from the nasopharynx or oropharynx.

2. Sample Preparation: The swab is then mixed with a buffer solution that helps to
release the viral or bacterial antigens from the swab.

3. Immunochromatographic Assay: The rapid antigen test kit contains a test strip with
immobilized antibodies that are specific to the viral or bacterial antigen being tested
for. The test strip has a region with immobilized antibodies and a detection region
with antibodies that are labeled with a colloidal gold conjugate or fluorescent dye.
4. Antigen-Antibody Binding: The sample is then applied to the test strip, and the
immobilized antibodies on the test strip bind to any viral or bacterial antigens that are
present in the sample.

5. Signal Production: If the viral or bacterial antigen is present, it binds to the labeled
antibodies in the detection region, producing a visible signal such as a color change or
fluorescent glow.

6. Result Interpretation: The healthcare provider interprets the results based on the
appearance of the signal. A positive result indicates that the viral or bacterial antigen
is present in the sample, while a negative result indicates that it is not.

7. The technical mechanism of action of rapid antigen tests is based on the principles of
immunochromatography, which is a type of lateral flow assay. The antibodies on the
test strip are designed to bind specifically to the viral or bacterial antigens, forming an

antigen-antibody complex. This complex is then detected by the labeled antibodies in

the detection region, producing a visible signal that can be interpreted by the
healthcare provider