based on microbiology 2nd year medical school

1. GENERAL VIROLOGY
Department of microbiology
and virology
of PIROGOV’s RSMU

2. Viruses and virions
• VIRUSES are obligate intracellular genetic
parasites that use the host cell’s machinery
for replication
• VIRUSES lack any cellular organization
• VIRUSES have one type of nucleic acid
(DNA or RNA), which, following the
adsorption of the virus into the host
cell, is then translated inside the host
cell to make viral proteins
• Viruses assemble new virus particles
within the host cell
• Virion is complete virus particle

3. Structure of viruses
• Virus contains a core of either DNA or RNA
surrounded by a capsid. Together, the nucleic acid
and the capsid is referred to as nucleocapsid
• Capsids are composed of smaller repetitive subunits
(capsomers). Capsomers are arranged in two
fundamental patterns of capsid structural
symmetry, icosahedral and helical
• Viruses with icosahedral symmetry contain a defined number
of structural subunits (20 triangular faces and 12 vertices).
HIV have a mixed symmetry: icosahedral in the
capsid, and helical in the nucleic acid core
•
Many viruses possess an envelope - enveloped
viruses; viruses without an envelope are called
nonenveloped viruses. The viral envelope is
composed of virus-specific proteins plus lipids and
carbohydrates derived from host cell membrane.

4. Enzymes
• Large number of viruses carry out virion-
associated enzymatic activities: RNA-
dependent RNA polymerase (RNA-
transcriptases) or a DNA-dependent RNA
polymerase
• Retroviruses contain an RNA-dependent DNA
polymerase known as reverse
transcriptase.

5. Classification of viruses
Basic principles
• The type and structure of the viral nucleic acid (single- or
double-stranded RNA or DNA)
• The presence or absence of an envelope
• The size
• The type of capsid symmetry
• The strategy for genome replication
• The viral antigens
• The route of transmission
• Epidemiologic features (susceptible hosts)

6. Classification of viruses
enveloped viruses nonenveloped viruses
Double-stranded DNA Double-stranded DNA
Single-stranded RNA
Single-stranded RNA
Single-stranded RNA
Double-stranded RNA
enveloped viruses nonenveloped viruses

7. Differences in size of typical
biological objects

9. DNA viruses, double-stranded
• Adenoviruses (Adenoviridae), nonenveloped,
icosahedral symmetry,
• Herpes simplex virus (Herpesviridae),
enveloped, icosahedral symmetry
• Poxvirus, vaccinia (Poxviridae), enveloped,
• Hepatitis B (Hepadnaviridae), enveloped
(mixed strandedness: 1 strand = party DNA)

10. Adenovirus

11. Herpesvirus

12. Poxvirus

13. Hepatitis B virus

14. RNA viruses
RNA viruses, single-stranded
Poliovirus (family Picornaviridae), nonenveloped, icosahedral
capsid symmetry
Yellow fever virus (Flaviviridae), enveloped, icosahedral
symmetry
Influenza viruses (Orthomyxoviridae), enveloped, helical
symmetry
Rabies virus (Rhabdoviridae), enveloped, helical symmetry
HIV (Retroviridae), enveloped, icosahedral capsid and helical
nucleocapsid
RNA viruses, double-stranded
Rotaviruses

15. Measles virus

16. Rubella virus

17. Influenza virus (RNA)

18. Influenza virus

19. Rabies virus (RNA)

20. HIV

21. HIV

22. Virus-cell interaction
• Once a virus reaches its target organs, it must then infect
and successfully replicate in host cells. Three possible
outcomes follow infection of a host cell by virus:
lytic infection, latent infection, or chronic infection
• In a lytic infection (polio, influenza viruses), the virus
undergoes multiple rounds of replication that results in
the death of the host cell
• A latent infection (Herpes simplex, retroviruses) does not
result in the immediate production of progenity virus. During cell growth, the
genome of the virus is replicated along with the chromosomes of the host cell.
Upon reactivation of the herpes simplex virus type 1, fever blisters or cold sores
result.
• A chronic (persistent) infection: virus particles
continue to be shed after the period of acute illness has
passed. This kind of infection is usually associated with RNA
viruses. Chronic infections are associated with a defective
host immunity
• Transformation of normal cells to tumor cells

24. The stages of viral-cell interaction
• I stage. Adsorption. The attachment of viruses to host cells
and specific binding of viral proteins to receptors on the host
cell surface
• II stage. Penetration. The viruses use different
strategies for penetration. Receptor-mediated endocytos,
for example. Adherence of the virus to clathrin-coated pits
and the gradual invagination of the membrane carrying the
virus into endosome
• III stage. Fusion of membranes. The viral envelope
fuses with the endosomal membrane
• IV stage. Uncoating: entry of the viral nucleic acid into the
cytoplasm
• V stage. Viral genome replication and macromolecular
synthesis. The synthesis requires the translation of viral
messenger RNA (mRNA)

25. VI stage of viral-cell interaction
• Assembly of virions and release from the host
cell
• Assembly of the nonenveloped and the nucleocapsid of
enveloped viruses proceed by the self-assembly of
viral capsomers into crystal-like arrays. Once the
capsid is formed, it becomes filled with the viral
nucleic acid to make a viable virion
• Nonenveloped virions are usually released when the
cell lyses.
• Enveloped viruses are typically released from infect
cells by budding. Virus-specified proteins inserted into
host cell membranes displace some of its normal
protein components, which results in the restructuring
of the membrane

27. Viral genome replication and protein synthesis

28. Viral replication
• The single-stranded positive polarity RNA
viruses
• The genomes of picornaviruses (Polio) and
togaviruses are said to have positive (+) strand
polarity: the nucleic acid of the virion to function
directly as mRNA.
• The cellular ribosomes bind to the mRNA to form large
polyribosomes that produce a single polyprotein. This
precursor molecule is then cleaved in a series of
proteolytic steps to produce the proteins of the core and
the capsid
• RNA polymerase known as transcriptase
synthesized a complementary (-) strand RNA using
the genomic RNA as template

29. The single-stranded, RNA+

30. The single-stranded negative polarity
RNA viruses (RNA-)
• The RNA of negative-strand viruses (Measles virus,
Rabies virus) does not carry coding sequences for
protein: these viruses synthesize mRNA by transcription
of genomic RNA. The genome is replicated via a (+) single-
stranded RNA intermediate
• RNA-containing, negative polarity Influenza viruses have
segmented genomes consisting of more than one RNA
molecule. RNA replication result in a unique mRNA for each
viral protein. Replication in the nucleus.

31. HIV
• These RNA viruses contain (+) single-stranded RNA but
employ a unique replicative strategy using a DNA
intermediate
• Viral RNA serves as a template for a virion RNA-
dependent DNA polymerase (reverse transcriptase).
The DNA is then integrated into host chromosomal
DNA

32. DNA viruses
• In cells infected with adenoviruses and
herpesviruses transcription of viral DNA in to mRNA
occurs in the nucleus of the host cell
• The first viral proteins produced after infection are
called early proteins. mRNAs encoding the capsid
polypeptides (late proteins) are transcribed

33. Hepatitis B virus
• The structure of HBV DNA is unique: it is a partly double-
stranded circular molecule
• The ‘minus’ strand (noncoding) is nicked and a
polymerase molecule is attached to its 5’ end
• The ‘plus’ strand contains a short RNA oligonucleotide at
its 5’ end and is shortened at its 3’ end
• Thus, the circular DNA genome has a single-stranded gap
• HBV travels to the liver. After uncoating viral genome is
converted into a fully double-stranded
partial ds DNA >ssRNA
Viral RNA is used as a template for reverse transcription,
resulting in the formation of viral DNA
ss RNA > ssDNA> dsDNA

34. Cultivation of viruses
Isolation of virus from clinical specimens is
done in
• cell cultures,
• embryonated eggs,
• animals (such as suckling mice, monkeys,
rabbits).
• Cell culture techniques involve the use of primary
cultures of cells prepared from organs of freshly
killed animals (e.g. monkey kidney cells); of
human diploid cell lines; and continuous
(heterodiploid) cell lines such as HeLa, Hep-2,
BHK-21, and Vero

35. Structure of embryonated
chicken egg

36. Cultivation of viruses
Example
• Inoculation into the amniotic cavity or the
allantoic cavity of embryonated chicken eggs
is useful for the isolation of influenza virus

37. Indication of viruses
• Indication of viruses can be achieved by
cytopathic effect (CPE), plaque assay, color
probe, hemagglutination and hemabsorbtion
• E.g., Orto- and paramyxoviruses (influenza,
parainfluenza, measles, mumps) may be
detected by the ability of infected cultures
to adsorb erythrocytes of animals
(hemadsorption)

38. Identification of viruses
• Identification of viruses can be achieved by
neutralization cytopathic effect (CPE), plaque assay,
color probe, hemagglutination and hemadsorbtion
• Examples
• Once cell cultures have been inoculated, the specimens are examined
for distinctive patterns of cytopathic effect (CPE)
. Herpes simplex virus and many enteroviruses produce early CPE,
whereas CPE due to CMV, rubella, and some adenoviruses may take
weeks
• Cultured cells are examined for cell lysis and vacuolization.
• The presence of syncytium suggest HSV, respiratory syncytial virus,
measles, or mumps virus.
• Cytomegaly is seen with HSV, varicella-zoster virus, and CMV.
• Immunocytochemical staining of cell cultures to detect viral antigens using
fluorescein or enzyme-conjugated specific antiviral antibodies may
aid in the detection and identification of many viruses.

39. Development and progression of viral cytopathology
Human embryo skin muscle cells were infected with human cytomegalovirus and
stained at selected times to demonstrate (A) uninfected cells, (B) late virus
cytopathic effects (nuclear inclusions, cell enlargement), (C) cell degeneration,
and (D) a focus of infected cells in a cell monolayer (i.e., a plaque).

40. Inclusions caused by different viruses

41. Respiratory syncytial virus.
Paramyxoviridae

42. Formation of multinucleated cells
The figure represents the cytopathology of measles virus-
induced syncytia.

43. Methods of diagnosis
for viral infections
• 1. Viroscopical method (microscopy)
• 2. Virological method means isolation and
identification of viruses in cell cultures and
embryonated eggs
• 3. Biological method uses laboratory animals
for isolation and identification of viruses
• 4. Serological method
• 5. Genetic-engineering method (PCR,
molecular hybridization)

44. Identification of viruses by specific antisera
• In some cases, examination of specimens by
immune electron microscopy is of diagnostic
value. The use of specific antisera to aggregate
virus in prepared stool specimens facilitates
electron microscopy detection of rotaviruses,
hepatitis A virus
• A 4-fold or greater increase in the antibody titer
to a specific viral agent in a patient’s acute and
convalescent (3 to 4 weeks later) sera is usually
considered diagnostic of acute infection
• A number of different types of antibodies including
neutralizing, complement-fixing, and hemagglutination-
inhibiting antibodies are routinely assayed.

45. PCR
• Hybridization and polymerase
chain reaction technique may
enable the detection of even
single copies of virus genomes in
tissue samples or cells from body
fluids

46. Mechanism of PCR

47. Antiviral immunity (NK-cells)
• Cytotoxity by natural killer (NK) cells provides
one of the earliest host defenses against viral
infection (peak activity at 2 to 3 day) and
precedes the appearance of antibody (7 days).
Natural killer cells are large granular lymphocytes
that bind to infected cells and then secrete
cytotoxic molecules –perforins and granzymes
• These cells are activated by virus-induced
interferons.

49. Apoptosis (action of NK-cells)

50. Antiviral immunity (CTLs)
• Cytotoxic T lymphocytes (CTLs) constitute a
specific virus-induced immune response
• CTLs (CD8+) recognize protein fragments of viruses
with major histocompatibility complex (MHC) antigens
• CTLs product granzymes ( action via apoptosis)

53. The antibody response
• The specific antibodies (production of СD4+
cells) do not usually play a primary role in terminating
acute viral infections but are very important in
preventing reinfection.
• Antibodies that protect the host by destroying the
infectivity of virus are called neutralizing
antibodies
• Neutralizing antibodies reduce viral infectivity by possibly
inhibiting attachment, penetration, or uncoating of virus. In
addition, such antibodies may produce aggregation of
virions, accelerate viral degradation

54. Structure of IgG

55. Interferons
• Interferons inhibit viral replication indirectly by inducing
the synthesis of cellular proteins that minimize viral
replication.
• There are three main kinds of interferons, called
α- , β-, γ-

57. IFNα

58. IFNβ

59. IFNγ