nptel – biotechnology – microbiology module 9 – the … · cytoplasm, or plasma membrane ......

NPTEL – Biotechnology – Microbiology

Joint initiative of IITs and IISc – Funded by MHRD of 21

Module 9 – The Viruses

Lecture 1: Viruses- Introduction and General Characteristics

What are Viruses?

Infectious acellular agents i.e. devoid of cell components like nucleus, organelles,

cytoplasm, or plasma membrane

Replicate or multiply only inside living host cell

Hence, also called obligate intracellular parasites

Possesses only one type of nucleic acids- either DNA or RNA but never both

(exception is cytomegalovirus)

Fig. 1.Virions of mimivirus, one of the largestvirusesand a parvovirus (arrowed), one of the smallest viruses.

History of Virology

Table 1.1 Selected Milestones in the History of Virology

Date Discovery

1892 Description of filterable infectious agent (TMV) (Ivanovsky)

1898 Concept of the virus as a contagious living form (TMV) (Beijerinck);

First description of an animal virus (FMDV) (Loeffler, Frosch)

1901 First identification of avian influenza; fowl plague virus (Lode, Gruber)

1901 First description of a human virus (yellow fever virus) (Reed et al.)

1903 Rabies virus identified (Remlinger, Riffat-Bay); Rabies inclusion bodies

described (Negri)

1908 Leukemia-causing virus identified (Ellerman, Bang)



1909 Poliovirus identified (Landsteiner, Popper)

1911 Discovery of solid tumor virus (RSV) (Rous)

1913 An early example of virus propagation in tissue culture (Steinhardt)

1915 First description of bacterial viruses (bacteriophages) (Twort, d'Herelle)

1931 Virus propagation in embryonated chicken eggs (Woodruff, Goodpasture)

1931 Use of mice as a host for viruses (Furth)

1931 Identification of swine influenza virus (Shope)

1933 Identification of human influenza virus (Smith et al.)

1933 Identification of rabbit papillomavirus (Shope)

1935 Tobacco mosaic virus crystallized (Stanley)

1936 Induction of carcinomas in other species by rabbit papillomavirus (Rous; Beard)

1938 Yellow fever vaccine (Thieler)

1939 One-step growth cycle for phages (Ellis, DelbrÃ¼ck)

1941 Recognition of influenza virus hemagglutination (HA); Discovery of first virus-

associated (receptor destroying enzyme, neuraminidase) enzyme (Hirst)

1947 Mutation and DNA repair in bacteriophages (multiplicity reactivation) (Luria)

1948 Poliovirus replication in non-neuronal cell culture (Enders, Weller, Robbins)

1952 Poliovirus plaque assay (Dulbecco)

1952 Bacteriophage genome is nucleic acid (Hershey, Chase)

1954 Polio vaccine developed (Salk)

1957 In vitro assembly of virus (TMV) (Fraenkel-Conrat, Williams)

1958 Bacteriophage lambda regulation paradigm (Pardee, Jacob, Monod)

1961 Discovery of messenger RNA (mRNA) using bacteriophages (Brenner, Jacob,

Meselson)

1961 Elucidation of the triplet code by genetic analysis of bacteriophages (Crick et

al.)

1961 Genetic definition of nonsense codons as stop signals for translation in

bacteriophages (Campbell, Epstein, Bernstein)

1963 Discovery of hepatitis B virus (Blumberg)

1964 Colinearity of a bacteriophage gene with the polypeptide chain (Sarabhai,

Stretton, Brenner); Discovery of first human tumor virus, EBV (Epstein-Barr)



1966 Pathways of macromolecular assembly of bacteriophages (Edgar, Wood)

1967 Phage Î» repressor isolated (Ptashne)

1967 Description of viroids (Diener)

1970 Discovery of retroviral reverse transcriptase (Temin, Baltimore)

1972 Recombinant DNA technology phage Î» and SV40 (Berg)

1973 Discovery that major histocompatibility complex (MHC) presents viral antigens

to lymphocytes (Doherty, Zinkernagel); First restriction enzyme map of a viral

genome, SV40 (Nathans)

1974 Phage lambda viral vectors for recombinant DNA technology (Murray, Davis,

Blattner, Enquist)

1976 Retroviral oncogenes are derived from cells (Bishop, Varmus)

1977 RNA splicing discovered in adenovirus (Roberts, Sharp, Chow, Broker)

1978 First viral genome sequenced (phiX174, Sanger)

1978 Virus crystal structure (TBSV) (Harrison)

1979 Discovery of the p53 tumor suppressor protein bound to the simian vacuolating

virus 40 (SV40) T-antigen (Levine, Lane)

1979 World Health Organization declares smallpox eradicated

1981 Development of infectious recombinant clone for an RNA virus, poliovirus

(Racaniello, Baltimore)

1981 Structure of first viral envelope protein (Wiley, Skehel, Wilson)

1983 Description of human immunodeficiency virus (HIV) as causative agent of

acquired immunodeficiency syndrome (AIDS) (Montagnier, Gallo)

1989 Hepatitis C virus cloned (Houghton et al.)

1990 Human gene therapy with a retrovirus vector

1994 Kaposi's sarcoma herpesvirus discovered (HHV-8) (Chang, Moore)

1997 HAART treatment for AIDS

2003 Severe acute respiratory syndrome (SARS) outbreak and containment; rapid

identification of novel human coronavirus

2005 Hepatitis C virus propagated in tissue culture (Chisari; Rice; Wakita)

2005 1918 influenza virus genome reconstructed and sequenced (Palese, Tumpey,

Taubenberger)



2006 Vaccine against human papillomavirus

2007 End of vaccination program for rinderpest

2011 Declaration of the eradication of rinderpest

TMV: tobacco mosaic virus; FMDV: foot and mouth disease virus; RSV: respiratory syncytial virus; TBSV:tomato bushy stunt virus; HAART: highly active anti-retroviral therapy.

General Characteristics of Viruses

1. Viral structure:Typical viral components are shown in Fig. 2. These components are a nucleic acid core and a surrounding protein coat called a capsid. In addition some viruses have a surrounding lipid bilayer membrane called an envelope.

Fig. 2.The components of helical virus

A. Nucleic acid

Viral genomes are either DNA or RNA (not both)

Nucleic acid may be single- or double-stranded

Fig. 3.Types of virus genomes



B. Capsid

protein coat

Protection of Nucleic Acid

Provides Specificity for Attachment

Capsomeres are subunits of the capsid

Fig. 4.Capsid structure

C. Envelope

Outer covering of some viruses

Envelope is derived from the host cell plasma membrane when the

virus buds out

Some enveloped viruses have spikes, which are viral glycoproteins

that project from the envelope

Naked (non-enveloped) viruses are protected by their capsid alone

Fig. 5.Enveloped helical virus



2. Size of viruses:

Determined by electron microscopy

Ranges from 20 to 14000 nm in length

Fig. 6. Size of different viruses



3. Shape of viruses:

Four basic morphologies

Icosahedral - efficient means to conserve and enclose space;

formcapsomers (planar faces formed by association of proteins)

Helical - capsid is shaped like a hollow protein tube

Enveloped - outer covering derived from the host cell’s nuclear or

plasmamembrane and often possessing spikes or peplomer projections

involved inattachment and entry into a host cell sometimes via their

enzymatic activity

Complex symmetry - viruses that fit neither of the above categories or

whichmay employ portions in combination, e.g., bacteriophage

Fig. 7.Types of viral symmetry

4. Host Range: The specific types of cells a virus can infect in its host species represent the host range of the virus. Animal virus

Plant virus

Bacterial virus (bacteriophage)

Host range is determined by attachment sites (receptors)



Important points to remember:

• VIRION – a complete single viral particle

• Obligatory intracellular parasites

• Contain DNA or RNA

• Do not undergo binary fission

• Sensitive to interferon

• Contain a protein coat

• Some are enclosed by an envelope

• Some viruses have spikes

• Most viruses infect only specific types of cells in one host

• Host range is determined by specific host attachment sites and cellular factors

(receptors)

• Viruses replicate through replication of their nucleic acid and synthesis of the

viral protein.

• Viruses do not multiply in chemically defined media

• All ss-RNA viruses with negative polarity have the enzyme transcriptase (RNA

dependent RNA polymerase) inside virions.

• Retroviruses and hepatitis B virus contain the enzyme reverse transcriptase.



REFERENCES:

Text Books:

1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).

Jones and Bartlett Student edition.

2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An

Introduction. Benjamin Cummings.

Reference Books:

1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw

Hill companies.

2. Biology, Raven and Jhonson, 6th edition (2001)

3. Microbiology, Pelczar. M.J , Chan E.C.S, Kreig N.R, 5th edition (2007)




Lecture 2 - The Bacteriophages What are Bacteriophages?

Bacteriophages are obligate intracellular parasite on bacteria that uses bacterial

machinery system for its own multiplication and development. These are commonly

referred as “phage”. Bacteriophages were jointly discovered by Frederick Twort (1915)

in England and by Felix d'Herelle (1917) at the Pasteur Institute in France.

“Bacteriophage” term was coined by Felix d'Herelle. Some of the examples of

bacteriophages are, Spherical phages such as φX174 (ssDNA), Filamentous phages such

as M13(ssDNA), T-even phages such as T2, T4 and T6 that infect E.coli, Temperate

phages such as λ and μ.

Fig. 8. Basic structure of Bacteriophages

Composition:

All bacteriophages contain nucleic acid as genetic material and protein.

Depending upon the phage, the genetic material may be either DNA or RNA. Certain

unusual modified bases are present in the genetic material of phages which protect the

phage genetic material from nucleases during infection. Protein surrounds the genetic

materials and protects to the surrounding environment.



Structure:

The basic structural features of T4 bacteriophages are illustrated in Figure 2. It is

approximately 200 nm long and 80-100 nm wide. Size of other phages is of 20 – 200nm

in length. All bacteriophages contain head and tail part. Head part is also termed ad

capsid which composed of one or different types of proteins. Genetic materials are

present inside and protected by capsid. Tails are attached to the capsid in most of the

phages. These are hollow tube like structure through which viruses inject their genetic

material inside the host during infection. Tail part is more complex structure in phages. In

T4 phage, tail part is surrounded by a contractile sheath and basal plate like structure

present at the end of tail from which certain tail fibres are attached. Tail fibres help in

attachment phages to bacteria and contractile sheath helps in contraction during infection.

Some of the phagesdo not contain tail fibres at the end. Certain other structures are

involved in these phages for binding to the bacterium during infection.

Fig. 9. Structure of T4 Bacteriophage

Infection of Host Cells:

The first step in the infections is binding of phage to bacterium which is mediated

by tail fibres are some other structures on those phages that lack tail fibres. Binding of

phage tail fibre to bacterium is through adsorption process and it is reversible. There are

specific receptors are present on bacterial cell surface through which phages bind on it by

its tail fibre. These receptors are proteins, lipopolysaccharides, pili and lipoproteins of



bacterium. Some phages lacking basal plate and tail fibre bind tightly to bacterial cell

surface and it is reversible.

After binding of phage to bacterium, there is contraction in tail by contractile

sheath and phages inject their genetic material through hollow tube like tail. Some phages

also contain certain enzymes that digest the bacterial cell surface. Phages that don’t

contain tail fibre and contractile sheath, uses different mechanism for inject its own

genetic material inside the host. Only genetic material of phages enters inside the

bacterium and the remainder of phages (ex. capsid) remain outside of bacterium.

Life Cycle:

There are two different types of life cycle present in phages: (i) Lytic cycle and

(ii) Lysogenic cycle. Lytic (virulent) cycle kill the host cell that they infect, while

lysogenic (temperate) cycle establishes a persistent infection without killing the host cell.

(A.) Lytic Cycle:

These are also known as virulent cycle because phages multiply inside the host

and lyse the cell at the end of its life cycle.After attachment of tail fibre to host, genetic

materials are injected inside the host. The time period between the entry of genetic

material inside the host and release of mature phage after end of life cycle is termed as

eclipse period. Synthesis of phage components and its packaging into mature phages

takes place in this period. After infection, the genetic material of phages uses host

biosynthetic machinery for replication, transcription and translation. Structural proteins

of phages (capsid, tail etc.) are also synthesized inside the host using host biosynthetic

machinery. After synthesis, genetic materials are packed inside the capsid and tail is

attached on it. This process is called as maturation of phages. In lytic phage, phages also

synthesized lysis protein. Bacterial cells are lysed due to accumulation of phage lysis

protein and mature phages are released into the medium. Around 10-1000 phages are

released from the bacterial cell. The average yield of phages per infected bacterial cell is

known as burst size.



Fig. 10. Lytic cycle of bacteriophage infection

(B.) Lysogenic cycle:

These are also known as temperate phage because phages multiply via the lytic

cycle or may enter a dormant state inside the cell. After entry of genetic material into the

host cell, the phage DNA integrates into the host chromosome and starts replication along

with it and passed to the daughter cells of host. Integration of phage DNA to host

chromosome is termed as prophage and bacteria is termed as lysogenic bacteria. Due to

integration of phage DNA to host chromosome, extra genes carried by phage get

expressed in the host cell and it may change the properties of bacterial cell. This process

is termed as lysogenisc or phage conversion.

Due to exposure to UV rays, ionizing radiations, mutagenic chemicals etc, DNA of phage

is released from host chromosome and separated phage DNA initiates lytic cycle

resulting in the lysis of cell and release of phages into the medium.



Significance of bacteriophages:

New characteristics are acquired using lysogenic conversion

Insertional mutation can be induced in bacterial chromosome by random insertion

of genes or nucleotides.

Latent infection in mammalian cells by retroviruses can be studied using Lambda

phage as model system.

In genetic engineering, phages are used extensively where they serve as cloning

vectors.

Phages are used to maintain libraries of genes and monoclonal antibodies

Natural removal of bacteria from water bodies can be done using bacteriophages

REFERENCES:

Text Books:





Reference Books:


Hill companies.






Lecture 3 -The Viruses of Eukaryotes

1. Characteristics of Eukaryotic Viruses

Obligate intracellular parasites

Infect and reproduce only within the living eukaryotic cells

Viruses contain single or double stranded DNA or RNA as their genomes (Fig. 1)

ssRNA able to function as mRNA is referred to as positive (+) sense or plus

strand RNA and if it is the equivalent to antisense RNA it said to be as minus

strand or negative (-) sense RNA

In certain cases, the genome encodes mRNAs which are of either sense

Processes that are found both in eukaryotes and their viruses- Glycosylation, RNA

processing and protein modification (proteolytic cleavage)

Fig. 11. General structure of enveloped Eukaryotic Virus

2. Structure of Eukaryotic viruses

The viral genome is enclosed by a protein coat known as capsid.

Capsid composed of protein subunits known as capsomeres

The nucleic acid genome along with the protective protein coat is called the

nucleocapsid.

Nucleocapsid can be of following symmetry:

Icosahedral,

Helical or

Enveloped symmetry



Majority of viruses have helical or icosahedral symmetry

(a.) Icosahedral symmetry

Icosahedral is regular polyhedran with 20 equivalent triangular faces 12 vertices.

In the icosahedral structure, the individual polypeptide molecules form a

geometrical structure that surrounds the nucleic acid

For example:Adenovirus has icosahedral structure (Fig. 2)

Fig. 12. Structure of Adenovirus



(b.) Helical symmetry

In viruses with helical symmetry, the polypeptide units are arranged as a helix

and form a rod like structure surrounding the nucleic acid genome

For example: TMV (Tobacco Mosaic Virus) has helical symmetry (Fig. 3)

Fig. 13. Structure of Tobacco Mosaic Virus

(c.) Enveloped symmetry

In envelope viruses,nucleocapsid is surrounded by a lipid bilayer and

glycoprotein derived from the modified host cell membrane called envelope.

Enveloped viruses are readily infectious agents if the envelope remains intact.

For example: Influenzavirus has enveloped symmetry

Fig. 14. Structure of Influenzavirus



4. Classification of Eukaryotic Viruses on the basis of Nucleic Acid

Major groups of viruses are distinguished on the basis of their nucleic acid content as:

DNA Viruses

RNA Viruses

Subsequent subdivisions are based largely on other properties of nucleic acids. The RNA

viruses can be ssRNA or dsRNA, although most are ssRNA.

(A.) DNA Viruses

Important Characteristics:

DNA as its genetic material

Replicationoccurs in the nucleus using a DNA-dependent DNA polymerase

DNA can be ssDNA or dsDNA and may be linear or circular

Important groups of DNA viruses are:

Adenoviridae

Herpesviridae

Poxviridae

Papovaviridae

Hepadnaviridae

Parvoviridae

The dsDNA viruses are further separated in families on the basis of the shape of their

DNA (linear or circular), their capsid shape and the presence or absence of an

envelope. Only one family of viruses has ssDNA (as shown in Table1)

Table 2: Classification of Eukaryotic DNA Viruses

Family Linear or circular DNA

Enveloped or naked

Capsid Shape

Typical size (nm)

Example

Double-Stranded DNA Viruses

Adenoviridae Linear Naked Polyhedral 75 Human adenoviruses

Herpesviridae Linear Enveloped Polyhedral 120-200 Simplexvirus

Poxviridae Linear Enveloped Complex 230×270 Orthopoxvirus

Papovaviridae Circular Naked Polyhedral 45-55 Human Papillomaviruses



Hepadnaviridae Circular Enveloped Polyhedral 40-45 Hepatitis B virus

Single-Stranded DNA Viruses

Parvoviridae Linear Naked Polyhedral 22 B19

(B.) RNA viruses

Important Characteristics:

RNA as their genetic material

Genome may be ssRNA or dsRNA

ssRNA viruses may be of positive (+) sense or negative (-) sense

Replicationoccurs in the cytoplasm using a RNA-dependent RNA polymerase

RNA dependent RNA polymerases are not having proofreading activity and hence

replicate their templates with a higher error rate.

Positive (+) sense viral RNA is similar to mRNA and thus can be immediately translated

by the host cell. Flaviviruses,togaviruses, poliovirus are some examples of (+) sense

RNA viruses.

Negative (-) sense viral RNAis complementary to mRNA and thus must be converted to

positive (+) sense RNA by an RNA dependent RNA polymerase before translation.

Influenza virus, Measles virus, Rabies virus are some examples of negative (-) sense

RNA viruses.

Double-stranded RNA viruses need to package an RNA dependent RNA polymerase to

make their mRNA after infection of the host cell. Examples of dsRNA viruses are

Rotaviruses which belong to family Reoviruses.

Important groups of RNA viruses are:

Picornaviridae

Togaviridae

Flaviviridae

Retroviridae

Paramyxoviridae

Orthomyxoviridae

Bunyaviridae

Reoviridae



The different families of RNA viruses are distinguished from one another by their nucleic

acid content, their capsid shape and presence or absence of an envelope (as shown in

Table 2)

Table 3: Classification of Eukaryotic RNA Viruses

Family No. of Copies

Enveloped or naked

Capsid Shape

Typical size (nm)

Example

(+) Sense RNA Viruses

Picornaviridae 1 Naked Polyhedral 18-30 Enterovirus

Togaviridae 1 Enveloped Polyhedral 40-90 Rubella virus

Flaviviridae 1 Enveloped Polyhedral 40-90 Flavivirus

Retroviridae 2 Enveloped Spherical 100 HTLV-I

(-) Sense RNA Viruses

Paramyxoviridae 1 Enveloped Helical 150-200 Morbillivirus

Rhabdoviridae 1 Enveloped Helical 70-180 Lyssavirus

Orthomyxoviridae 1 copy in 8 segments

Enveloped Helical 100-200 Influenzavirus

Filoviridae 1 Enveloped Filamentous 80 Filovirus

Bunyaviridae 1 copy in 3 segments

Enveloped Spherical 90-120 Hantavirus

Double-Stranded RNA Viruses

Reoviridae 1 copy in 10-12 segments

Naked Polyhedral 70 Rotavirus



REFERENCES:

Text Books:





Reference Books:


Hill companies.



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