Microbiology 2

Virology

Virology
▪ study of viruses

Viruses
▪ smallest infectious agents that can pass through filters
▪ Obligate intracellular parasite
▪ Are infectious agents with both living and non living characteristics
▪ All viruses are made up of a core of genetic material
▪ Lengths are measured in millions of a millimetre; nanometer (20 -200 nm)
▪ Electron Microscopy
▪ Culture: cell line or tissue culture

Viral Structure
▪ Viruses cannot even reproduce by themselves

Capsid protective protein coat either helical or icosahedral

Capsomeres subunits of capsid which consist of several identical or different protein molecule

Nucleocapsid capsid together with the enclosed nucleic acid

Nucleic acid core constitutes genetic material or viral genome
Envelope lipid derived from the host cell

Spikes glycoprotein molecule that are easily visible in electron microscope

RNA/ DNA nucleic acid genome

Virion
Functions:
▪ To protect the viral genome from destructive agents in external environment.
▪ To introduce the viral genome into the host cell.

Criteria for Virus Classification

I. Nucleic Acid Composition
▪ DNA / RNA
▪ Single stranded – may be of positive RNA (mRNA) or negative anti-mRNA polarity
▪ Double stranded
II. Capsid Morphology
▪ Helical
▪ Icosahedral
▪ Complex
III. Envelope
▪ Presence or absence

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All faces of icosahedron are identical

nucleic acid inside the capsid

A. Helical Symmetry
o Non-enveloped plant virus, tobacco mosaic virus
o Enveloped, helically symmetrical viruses (influenza, rabies viruses) the capsid is more flexible and
longer and appears negative stain rather like a telephone cord

B. Complex Symmetry
o These are regular structures but the nature of the symmetry is not fully understood.

*Pox virus seen by negative staining

Five Basic Structural Forms of Viruses

1. Naked Icosahedral
o poliovirus, adenovirus, Hepatitis A virus

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2. Naked Helical
o tobacco mosaic virus (no human virus with this structure are known)

3. Enveloped Icosahedral
o herpesvirus, yellow fever virus, rubella virus

4. Enveloped Helical
o rabies virus, influenza virus, parainfluenza virus, mumps, measles

5. Complex
o poxvirus

Unconventional Agents
➢ Main kinds that have been studied are Viroids and Prions

a. Viroids
o small (less than 400 nucleotides) single-stranded, circular RNAs

b. Prions
o contain protein only, small, proteinaceous particle, they may or may not contain nucleic acid but if
there is any, there is very little and not enough to code for protein
o Ex. Prion-caused human disease
o Kuru – degenerative disorder

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o Creutzfeldt Jacob disease – rare; memorly loss

o scrapie (sheep or goat) – rare degenerative neurological disorder
o Gerstmann-Straussler Scheinker syndrome (GSS) – cause dementia and ataxia
o Bovine spongiform encephalopathy – Mad Cow disease

ARE VIRUSES LIVING OR DEAD?

Classification of Viruses
o System of virus classification is based on structure and composition of virus particle

a. NUCLEIC ACID
✓ RNA/DNA
✓ Single-stranded or double-stranded
✓ Non-segmented or segmented
✓ Linear or circular
✓ Symmetry

b. VIRION STRUCTURE
✓ enveloped or not enveloped

Multiplication
1. The virus must recognize and attach to its host cell
2. Viruses are limited as to the type of host cell in which they can multiply
3. Viruses adsorb to their host cell surface via specific anti-receptor molecules
4. Penetration into the host cell is often energy dependent and may occur by 3 different mechanisms.
▪ Translocation of the plasma membrane
▪ Pinocytosis into cytoplasmic vacuoles
▪ fusion of the plasma membrane with the viral envelope
Non-enveloped viruses may enter via translocation or pinocytosis
Enveloped viruses typical enter via fusion.
5. Once inside the host cell, uncoating releases the viral genome to be replicated.

A. Primary Characteristic used in Classification

DNA VIRUSES

Double- stranded Single-stranded

Enveloped Non-Enveloped Complex-enveloped Non-enveloped

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Circular Linear
Herpesviridae Papilloviridae Adenoviridae Poxviridae Parvoviridae
Polyomaviridae

RNA VIRUSES

Single-stranded (positive sense) Single-stranded Double-stranded

(negative sense)

Enveloped Non-enveloped Enveloped Non-enveloped

Icosahedral Helical Icosahedral Helical Icosahedral
Flaviviridae Coronaviridae Picornaviridae Orthomyxoviridae Reoviridae
Togaviridae Caliciviridae Paramyxoviridae
Retroviridae Rhabdoviridae
Filoviridae
Bunyaviridae
Arenaviridae

B. Secondary Characteristic
o Replication strategy – mode of replication

EFFECT OF VIRUSES ON HOST

o Many viruses inhibit host RNA, DNA or protein synthesis.

o The mechanisms by which the virus does this vary widely.

a. Cytopathic effect(CPE)

▪ The presence of the virus often gives rise to morphological changes in the host cell
▪ . Cytopathic effects (CPE) may consist of cell rounding, disorientation, swelling or shrinking, death,
detachment from the surface, etc.
▪ Many viruses induce apoptosis in infected cells.
▪ This can be an important part of the host cell defense against a virus - cell death before the
completion of the viral replication cycle may limit the number of progeny and the spread of infection.
▪ Some viruses delay or prevent apoptosis - thus giving themselves a chance to replicate more virions.
▪ The cytopathic effects produced by different viruses depend on the virus and the cells on which it is
grown.

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Types of changes:

Ballooning/ giant cell - HSV Refractive – enterovirus

Grape-like – adenoviruses Round necrosis – rhinoviruses (survives
Hemadsorption – influenza, at 33C)
parainfluenza, mumpls, measles Syncytium – rubella, respiratory
No CPE – influenza, mumps, measles syncytial virus, measles

b. Assays for plaque-forming units

▪ The CPE effect can be used to quantitate infectious virus particles by the plaque-forming unit assay.
▪ Cells are grown on a flat surface until they form a monolayer of cells covering a plastic bottle or dish.
▪ They are then infected with the virus. Surrounding cells are infected by the newly replicated virus and
they too are killed.
▪ This process may repeat several times.
▪ The cells are then stained with a dye which stains only living cells.
▪ The dead cells in the plaque do not stain and appear as unstained areas on a colored background.
Each plaque is the result of infection of one cell by one virus followed by replication and spreading of
that virus.

DNA VIRUSES

Symmetry Envelope Size Disease/ Example

Parvoviridae Icosahedral - 20 nm -Human Parvo B19
Hepadnaviridae Icosahedral + 42 nm -Hepatitis B virus
Icosahedral - 40-60 nm -Warts
Papillomaviridae -HPV
Icosahedral - 40-60 nm -SV40
Polyomaviridae -BK Polyomavirus
Adenoviridae Icosahedral - 80 nm -JC polyomavirus
Icosahedral + 190 nm -Herpes simplex virus 1 & 2
-Varicella zoster virus
-Chickenpox
-Shingles
Herpesviridae
-Epstein Barr Virus
-Infectious Mononucleosis
-CMV

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Complex + 200 nm x 350 -Vaccinia

Poxviridae nm -Smallpox
-Cowpox viruses

RNA VIRUSES – Positive Sense

Symmetry Envelope Size Disease/Example

Icosahedral - 30 nm Enteroviruses
- Poliovirus
-Coxsakie virus
Picornaviridae -Hepatitis A virus
-Echovirus
Rhinoviruses
Icosahedral - 35 nm Norwalk virus
Caliiciviridae Hepatitis E virus
Icosahedral + 60-70 nm Rubella virus
-Western Equine Encephalitis
-Eastern Equine Encephalitis
Togaviridae -Venezuelan Equine Encephalitis
Chikungunya virus
-Sindbis virus
-Semliki Forest virus
Icosahedral + 40-55 nm -Yellow fever
-Dengue
Flaviviridae Japanese Encephalitis
-St. louise Encephalitis
Helical + 75-160 nm -Coronavirus
Coronaviridae -SARS
Icosahedral + 100 nm -HIV
Retroviridae -Human T-cell Leukemia

RNA VIRUSES – NEGATIVE SENSE

Symmetry Envelope Size Diseases/ Examples

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Helical + 60-180 nm -Rabies

Rhabdoviridae -Vesicular Stomatitis virus
-Mokola virus

Helical + 150-300 nm -Newcastle virus

Mumps
Paramyxoviridae Measles
-parainfluenza virus
-respiratory syncytial virus

Orthomyxoviridae Helical + 80-120 nm -Influenza type A, B, C virus

Helical + 95 nm -California encephalitis virus
-Lacrosse
-Crimean-Congo hemorrhagic fever
Bunyaviridae -Rift valley fever
-hanta virus genus (Korean
hemorrhagic fever)
Helical + 50-300 nm -lymphocytic choriomeningitis virus
-Junin virus
Arenaviridae
-Machupo virus

Helical + 80-1000 nm -Ebola virus

-Marburg virus
Filoviridae
-Reston virus

Icosahedral - 75 nm -Reovirus
-Rotavirus
Reoviridae
-Orbivirus

Rule: DNA Viruses

1) All are D-S DNA except Parvovirus
2) All are icosahedral except Poxvirus
3) All are enveloped except PAP
4) All multiply in nucleus except Poxvirus

Rule: RNA Viruses

1) All are S-S RNA except Reovirus
2) All are enveloped except PCR
3) All are non-segmented except ROBA
4) Generally helical except the (+) sense RNA viruses
5) (+) sense – Call Pico & Flo To Come Right
6) (-) sense – Pairing OF Rats at Bunny’s Area

Resistance of Viruses:
▪ Resistant to antimicrobials, antibacterials

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▪ Sodium hypochlorite, formaldehyde and dilute HCl are efficient

▪ Can survive long periods at -70C when freeze dried
▪ Killed with moist heat but are somewhat resistant to dry heat
▪ UV and Xray radiation inactivates viruses
▪ Chloroform and ether will inactivate viruses that have lipid envelope

Transmission of Viral diseases

▪ Droplet infection ▪ Direct Inoculation
▪ Ingestion ▪ Latent
▪ Direct contact ▪ Transplacental
▪ Insect-borne/arthropod-borne

Specimen Collection:
1. Specimens should be collected early in the acute phase of infection
2. Inoculation of specimens into tissue culture
3. Virus Transport Media
a. Modified Stuart’s medium
b. Modified Hank’s medium
c. Leibovitz-Emory medium
d. Buffered saline with protein as stabilizer and added antibiotics
e. Veil Infusion broth
f. 1% Bovine serum albumin
g. Skimmed Cow’s Milk
4. In transporting, specimens should be kept at 4C or on crushed ice until inoculated, however, if there is a
delay of more than 4 days, freezing the specimens at -70C is required.

Specimens to be collected

Clinical Syndromes Specimens Virus commonly associated

-throat swab -influenza
-nasal washings -parainfluenza
Respiratory syndrome -nasophrayngeal aspirate -adenovirus
-rhinovirus
-stool -rotavirus
-rectal swab -norwalk
Gastroenteritis -throat swab -adenovirus
-enterovirus

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-skin scrappings -Varicella zoster

-retal swab -Herpes simplex
Skin lesions or rash -veisuclar fluids or swabs -measles
-rubella
-enterovirus
-Brain biopsy -Herpes simples
Encephalitis or aseptic -Blood and CSF -Togavirus
meningitis -Throat and rectal swabs -Enterovirs
-Rabies virus
-stool -Hepatitis A and B
Hepatitis
-serum
-throat swab -rubella
Congenital infection -urine -megalovirus
-single serum (mother/infant)

Laboratory Diagnosis
A. Direct Microscopic Examination
▪ For detecting the presence of characteristic viral inclusions
▪ Commonly used for herpes simplex, herpes zoster and CMV detection

Rabies virus Negri bodies

Yellow fever virus Torres- Councilman bodies
Fowlpox virus Bollinger bodies
Variola and Varicella Guarnieri-Puschen bodies
Ectromelia Marshall bodies
Herpes simplex Papanicolau stain of cervical smears show giant cells and
intranuclear inclusions
Cytomegalovirus intranuclear inclusions stain with hematoxylin – eosin

Herpes zoster multinucleated giant cells and intranuclear inclusions seen

on Tzanek’s

B. Electron Microscope

C. Antigen Detection
1. Immunofluorescence Microscopy (FAT)
a. Direct
o uses fluorescence-labeled specific antibody

b. Indirect
o a fluorescence-labeled antiglobulin is used to detect the unlabelled specific antibody
2. Immunoperoxidase Staining

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3. Solid-Phase Immunoassay
a. RIA
b. ELISA
c. Particle agglutination

D.Animal Inoculation Test

D. Nucleic Acid Hybridization

F. Cultivation
1. Chick Embryo
a. sites of inoculation
o yolk sac, amniotic fluid, allantoic membrane, chorioallantoic membrane

b. signs of growth
o death of the embryo
▪ pock formation – red and white lesions usually seen in the chorioallantoic membrane
▪ development of hemagglutinins – determined by hemagglutination method embryonic fluids

2. Tissue Culture Media

a. Primary Monkey Kidney (PMK)
b. Human fetal diploid (HFD)
c. Hep-2 continuous cell line
d. HeLa cells
e. AGMK, vero cells, BHK, HEK, HEL

Type of Cell Culture:

a. Primary cell
b. Semi-continuous
c. Continuous

Signs of growth:
a. CPE
▪ evidence of host cell infection and damage or morphological changes in the cells due to viral
proliferation that can be readily observed in unfixed, unstained cell cultures

b. Plaque formations
▪ small zones of cell destruction or areas of clearness that can be seen with the naked eye

c. Production of hemagglutinins
▪ detected by hemadsorption/hemagglutination of guinea pig erythrocytes

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d. Foci formation
▪ many tumor viruses do not destroy cells but rather cause them to change morphology and multiply
at a faster rate (transformed cells)
▪ Colonies of transformed cells develop into foci that can be visualized with the naked eye

G. Serologic Tests
▪ CF, neutralization, ▪ CIE
▪ HI ▪ RI
▪ PHA ▪ ELISA
▪ IFA ▪ ACIF
▪ IAHA ▪ SRH
▪ IEM

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