VIRUSES

KEY CHARACTERISTICS!

1. Size: 20 to 300 nm; visualized

using electron microscope
2. Basic structural unit/infectious particle is called a
VIRION Capsid
Capsid

3. They basically consist of NUCLEIC ACID

CORE (RNA or DNA) and protein coat
called CAPSID
4. Some may have external lipid
ENVELOPE which covers the capsid.
The envelope may have virus-
encoded GLYCOPROTEIN SPIKES.
5. Some may have tail, sheath and fibers.
6. They DO NOT have ORGANELLES OR RIBOSOMES.
7. Viruses contain genetic material, but NOT
THE ENZYME NEEDED TO REPLICATE. Thus,
they cannot replicate on their own. In
order to do so, they need living host cells.
As such, viruses may be described as
OBLIGATE INTRACELLULAR PARASITES
8. Displays TROPISM – capability of a virus to
infect a distinct group of cells in the host.
Made possible because of the interaction
of viral surface structures (such as
glycoproteins) with receptors on the host
cell.
(Example: Rabies virus - CNS tissue; Influenza – respiratory epithelium)

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VIRAL MORPHOLOGY

1. Nucleic Acid– May be RNA or DNA. Just like in humans, it can encode hundreds of enzymes and
structural proteins. The strands of the nucleic acid may be:
a. single-stranded or double stranded,
b. linear (having two ends) or looped,
c. in separate segments or one continuous strand
RNA VIRUSES
TYPES OF RNA
(+) Positive-stranded - when virus enters the host cell, its RNA can be immediately translated by the host’s
ribosome into protein

(-) Negative-stranded - when virus enters the host cell, it cannot be immediately translated.
- it has to be transcribed first to a positive strand of RNA, by the help of the enzyme
RNA-dependent RNA polymerase (present in the viral capsid, and not present in
humans)

RNA of retroviruses - the RNA is transcribed in a reverse fashion (“retrograde”) into DNA, by the help of
the unique enzyme reverse transcriptase (present in virus)

DNA VIRUSES
- DNA cannot be translated directly into a protein; it has to be transcribed into mRNA first
-These viruses have both positive and negative strand. Positive strand is read, while negative strand
is ignored… . Positive strand is used as template for transcription into mRNA

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 2. Capsid – houses the genome
a. Capsomere – basic unit of capsid; globular protein subunits composed of polypeptide
chain that makes up the capsid
b. Nucleocapsid – Core + Capsid
c. Some may have STRUCTURAL PROTEINS and ENZYMES inside their capsid
TYPES
Icosahedral Helical
– capsomeres arranged in a triangle, and combined – individual capsomere is attached to RNA (always
with many triangles; 20 sides or facets RNA), and is coiled helically. Most are spherical except
for rhabdoviruses (rabies virus), which have bullet-
shaped capsid

3. Envelope – lipid bilayer membrane embedded with proteins or glycoprotein spikes. It is

acquired by virus from the host through budding or tearing off a part of the host cell’s
membrane as they leave the cell after viral replication.
a. ENVELOPED VIRUS - has envelope; susceptible to drying out and destruction in the
environment; ether-labile
i. Glycoprotein spikes – assist in attachment of virus to cells
ii. Matrix protein – found between envelope and capsid; has enzymatic activities
b. NAKED VIRUS – does not have envelope; very resistant; ether-stable

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VIRAL REPLICATION – (AKA infectious cycle)

STEPS: APUERLAR/APUMAR

1. Attachment (AKA Adsorption) – recognition and binding of viral proteins to receptors

on host cell
2. Penetration – virus enters the host cell through fusion (enveloped
viruses), endocytosis (naked viruses) or injection of viral nucleic acid
(bacteriophage).
a. Fusion of virus and target cell may also lead to fusion with other

Injection

Fusion Endocytosis

nearby cells, forming multinucleated cells called syncytia

3. Uncoating – once the virus is inside, the capsid dissociates or is degraded

by enzymes, leading to release of viral genomes into cytoplasm or nucleus
4. Early mRNA and protein synthesis – synthesis of nonstructural elements (e.g.
enzymes)
5. Replication – viral nucleic acid is replicated to provide genomes for progeny virions
6. Late mRNA and protein synthesis – synthesis of structural elements (e.g. capsid)
7. Assembly of Virion – nonstructural elements, genome and structural
elements are assembled into a virus particle.
8. Release of virions from cell
a. Naked viruses – released from cell through cell lysis  rapid death of
host cell
b. Enveloped viruses – released from cell through “budding.” During
this, the virus acquires the envelope by tearing off a part of the host’s
cell membrane.

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5
 DNA Viruses - replicate in the nucleus of the host cell

PaPaAd Po HeHe – Parvovirus, Papova viruses, Adenovirus, Poxvirus, Herpes virus, Hepadna viruses

DNA

Naked Envelope

Single-stranded Double-stranded Double-stranded

Icosahedral Icosahedral Complex Icosahedral

Parvovirus Papova Adeno Poxvirus Hepadna Herpes

Simple – only
Capsid is like a
has one strand.
box – POX in a
> ONE PAR hole
BOX; replicates
in golf in cytoplasm of

Remember!

All are dsDNA except: PARVO

All are icosahedral except POX
All of them replicate in nucleus except POX

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RNA Viruses - replicate in the cytoplasm of host cells
RNA

Single-stranded Double-stranded

Positive-stranded Negative-stranded Naked

Naked Enveloped Enveloped Reo

Picorna Flavi Bunya

Calici Toga Orthymyxo

Retro Arena

Corona Filo

Rhabdo

Paramyxo

Remember!
All are ssRNA except: REOVIRIDAE
3 Naked Families: Leonardo di CaPiReo (Calici, Picorna, Reo)
Most have helical capsid except these five icosahedrals:
di CaPiReo in a Yellow Toga (Calici, Picorna, Reo, Flavi, Toga)
Helical capsid but bullet—shaped – RHABDO
Replicates in nucleus – RETRO, ORTHOMYXO
Segmented, enveloped – BOAR (Bunya, Orthomyxo, Arena, Retro)

Prepared by: Julie Anne L. Nano, RMT 7

All pictures used in this document belong to their rightful owners.
 LECTURE NOTES ON MYCOLOGY
GENERAL CHARACTERISTICS OF FUNGI

MYCOLOGY- THE STUDY OF FUNGI

1. Fungi are Eukaryotic

- contains nucleus and mitochondria
2. Lack chlorophyll, thus heterotrophic
3. Cell walls are made of chitin
- Vs. Cellulose in Plants
4. Ergosterol present in the cell membrane
- Vs. Cholesterol in Animals
5. Grow best at neutral pH
6. Lack susceptibility to antibacterial agents

Hyphae – microscopic units of fungi

Mycelium – intertwining structure composed of hyphae

YEAST VS MOLDS

YEAST:
 Single Vegetative Cells, reproduce by budding or fission
 Colonies are creamy; bacteria like
MOLDS:
 Colonies are creamy; bacteria like
 Colonies are fuzzy
 May produce spores (mainly Conidia)
 TERMS:
o Septate: with crosswalls
o Aseptate/Coenocytic: no crosswalls
o Hyaline: no pigmentation
o Phaeoid/Dematiaceous: with pigmentation

Prepared by: Lee-an V. Anayon, RMT 1

 Diphasic – exists as mold in vitro (at both RT and body temp); yeast in vivo
Yeast 37 C In-Vivo Invasive Mold 22-25 C Outside Infective (Spores)
 Monophasic – exists in only one form (yeast or mold)
 Dimorphic – has two forms; depends on temperature
 Polymorphic – may exist as both yeast and mold in the same culture

TAXONOMY

 Mucorales – (Mucor, Rhizopus)

 Asexual reproduction: sporangiospores
 Ascomycota –( Microsporum spp., Trichophyton spp.)
 Sexual reproduction: Ascospores within ascus
 May also reproduce asexually
 Basidiomycota – (Cryptococcus neoformans )
 Reproduce by budding

SPORES
FOR REPRODUCTION:
 ASEXUAL SPORES
o Thallospores
o Blastospores – budding (Candida)
o Arthrospores – rectangular, thick walled (Coccidioides)
o Chlamydospores – spores surrounded by a thick wall (Candida)
o Conidia
o Macroconidia – large, multicelled, spindle shaped
o Microconidia – small, single celled
 SEXUAL SPORES
o Ascospores – develop within an ascus
o Basidiospores – develop within a club-shaped structure
o Oospores, Zygospores

LABORATORY DIAGNOSIS
SPECIMEN COLLECTION
 Skin, and Nail – scrapings; clean surface with 70% isopropyl alcohol before
scraping

Prepared by: Lee-an V. Anayon, RMT 2

 Hair – pluck with forceps
 For dermatophytes
 CSF – for meningitis
 Should not contain antibacterial or antifungal agents
 Concentrate using filter
 Blood, Bone Marrow
 Lysis centrifugation system to lyse WBCs and RBCs in order to release
microorganisms; centrifuged and inoculated on media

MICROSCOPIC METHODS

 Saline Mount – for direct examination of fungal elements

 10% Potassium Hydroxide (KOH) – to dissolve keratin in hair, skin, and nails
for easier examination; fungal elements appear refractile
o Alternative: NaOH
 India Ink – negative (background staining) for visualization of the capsule of
Cryptococcus neoformans
o  Yeast with clear halo against a black background
 Lactophenol Cotton Blue
o Lactic Acid preserves fungal structures
o Phenol kills organisms
o Cotton Blue stains the chitin in fungal cell walls
 Calcofluor White
o May be coupled with KOH to enhance sensitivity
o Fluorescent stain for fungi (requires fluorescent microscope)
o Binds to chitin in fungal cell walls  Yellow-green fluorescence

EMAXINATION TECNIQUES AND CULTURE MEDIA

1. Most fungi grow optimally at 30 C
2. Grow also at 37 C if the agent is dimorphic (most pathogens)
3. Maintain for 4-6 weeks
4. Grow molds in tubed media with screw-cap tops (not cotton) to minimize
airborne spread of spores

Prepared by: Lee-an V. Anayon, RMT 3

Microscopic Examination Techniques
 Tease Mount
 Cellophane Tape Preparation
 Slide Culture
o Fungus is grown on a cube of agar on a slide
o For optimal examination of the fungus without disturbing the
general structure
o Rapid and easy to perform

Culture Media
o Must contain a source of nitrogen (amino acids) and carbon (CHO)
o May be supplemented with Vitamins, Minerals, Indicators, and
Inhibitors
o May be poured on Petri Dishes (plated media) or on large tubes.
o Petri Dish: Pour thicker than bacterial media; seal to prevent drying
o Tubes: Screw cap (cotton plug is not acceptable) to prevent spread of
spores
 Inhibitors:
o Cycloheximide: Inhibits fungal contaminants (saprobes)
o Chloramphenicol: Inhibits gram  and gram negative bacterial
contaminants

MYCOSES
MYCOSIS-DISEASE CAUSED BY FUNGI
 MYCOSES- PLURAL FORM

1. CUTANEOUS MYCOSES
- Non-invasive; only infect hair, skin, and nails
- May be caused by Dermatophytes or Nondermatophytes

DERMATOPHYTES: TEM (Trichophyton, Epidermophyton, Microsporum)

- These organisms utilize keratin as nitrogen source
 Trichophyton – infect skin, hair, and nails
 Epidermophython – infect skin and hair (not nails)
 Microsporum – infect skin and nails (not hair)

Prepared by: Lee-an V. Anayon, RMT 4

 NON DERMATOPHYTES
 Malassezia furfur
 Exophiala werneckii
 Piedraia hortae
 Trichosporon beigelii

2. SUBCUTAENOUS MYCOSES
 Result from a traumatic skin puncture from thorns or vegetation
contaminated with fungi
 Commonly found in soil – geophilic
ORGANISMS INVOLVED:
SPMC:
Sporothrichosis
Phaeohyphomycosis
Mycetoma
Chromoblastomycosis

3. SYSTEMIC MYCOSES
- A type of mycoses that involves the lungs and other organs in the body

Dimorphic fungi; Possesses Thermal morphism

Yeast phase (37 C): Invasive phase
Mold phase (22-25 C, RT): Infective phase

 Mode of transmission: Inhalation of spores; - Originate primarily in

the lungs and may spread to many organ systems.
 Laboratory workers are in danger when handling the mold phase of fungi in
culture (Biosafety Level 3 due to spore production)
 ORGANISMS INVOLVED:
Blastomyces dermatitidis
Coccidioides immitis
Histoplasma capsulatum
Paracoccidioides brasiliensis

Prepared by: Lee-an V. Anayon, RMT 5

4. OPPORTUNISTIC MYCOSES
 Saprophytes in the environment
- Normal flora of the body or in environment
- Occurs in immunocompromised patient
 ORGANISM INVOLVED:
Candida
Aspergillus
Cryptococcus

 Candida spp.

Causative agent of: Candidiasis, Vulvovaginitis, Oral Thrush

 Members of the genus Candida:
1. C.albicans
2. C.parapsilosis
3. C. tropicalis
4. C. glabrata (or Torulopsis glabrata)
Infection may be Endogenous or Exogenous
 ENDOGENOUS: Lowering of host‟s integrity vs. Candida
(immunocompromised, mucosal irritation)
 EXOGENOUS: Candida infection from outside sources
(contamination)
Candida albicans may grow on BAP Colonies typically appear as white, „star-
shaped‟ colonies or with foot processes.
 DIAGNOSIS:
- Culture: Cornmeal agar (demonstration of chlamydospores)
- Carbohydrate assimilation and fermentation
GERM TUBE TEST
 For presumptive identification of C. albicans
 Uses serum, 37 C for 3 hours
 = germ tube formation
(-) = pseudohyphae / no germ tube (Control: C. tropicalis)

Germ tube: long tube with no constrictions

Pseudohyphae: with constriction at base

Prepared by: Lee-an V. Anayon, RMT 6

 Candida albicans (and other yeasts) will stain as gram positive and may
resemble cocci, but larger and more ovoid in shape (budding may be seen)

 Cryptococcus neoformans

Causative agent of: Cryptococcosis

 An encapsulated yeast
 Reproduces through budding
 DISEASE ASSOCIATION:
Fungal Meningitis, Cutaneous cryptococcosis, Pulmonary cryptococcosis
 DIAGNOSIS:
o Negative stain: India ink (demonstration of capsule)
o Gram stain: Characteristic "starburst classical pattern" on CSF
sample
o Nitrate reduction test: (+)
o Urease: (+)
o Latex Agglutination Test: Detection of cryptococcal Ag
Culture (these refer to the same medium)
o Birdseed agar / Nigerseed agar / Staib's Medium

These media detect Phenol Oxidase activity of C. neoformans

o Phenol Oxidase: (+) (black color or melanin production)

 Aspergillus spp
Causative agent: ASPERGILLOSIS
 aka Farmer‟s lung
 The most pervasive of all fungi
 Second most isolated fungus after Candida A.fumigatus (MOST
COMMON)

 DISEASE ASSOCIATION
 Usually secondary to serious illnesses such as leukemia, AID
 Aspergilloma (fungus ball) in tissues

Aflatoxin – toxin produced by A. flavus Found in contaminated food; has

hepatotoxic activity
Prepared by: Lee-an V. Anayon, RMT 7
  DIAGNOSIS:
Microscopy: Presence of large, branching, septate hyphae
Media: Czapek Agar

ADDITIONAL NOTES:
 Pneumocystis spp.
 Originally thought to be a protozoan, but is now classified as a fungus
 Pneumocystis carinii: rats
 P. jiroveci: humans
Opportunistic infection in AIDS patients Associated with PJP/PCP (Fatal
Pneumonia)
 Non-Culturable!
 No Mold/Yeast form
 Trophozoite / Cyst forms instead
 LAB DIAGNOSIS:
o Specimen: BAL, Respiratory spx
o Staining: Silver Stain

Prepared by: Lee-an V. Anayon, RMT 8