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common because of the large number of different causative viruses, and reinfections may occur. Bacterial

agents associated with rhinitis (10%-15%) include Chlamydia pneumoniae, Mycoplasma

pneumoniae, and Group A streptococci.

(Table 1): Viral Agents That Can Cause Rhinitis:

Rhinoviruses

Coronaviruses

Adenoviruses

Parainfluenza and influenza viruses

Respiratory syncytial virus

Enterovirus

Miscellaneous Infections Caused by Other Agents:

Corynebacterium diphtheriae. Pharyngitis caused by Corynebacterium diphtheriae is less common than

streptococcal pharyngitis. After an incubation period of 2 to 4 days, diphtheria usually presents as pharyngitis

or tonsillitis.

Patients are often febrile and complain of sore throat and malaise (body discomfort). The hallmark for

diphtheria is the presence of an exudate or membrane that is usually on the tonsils or pharyngeal wall. The

graywhite

membrane is a result of the action of diphtheria toxin on the epithelium at the site of infection. Complications

occur frequently with diphtheria and are usually seen during the last stage of the disease (paroxysmal stage).

The most feared complications are those involving the central nervous system such as seizures, coma, or

blindness.

Bordetella pertussis. Although mass immunization programs have greatly reduced the incidence of pertussis,

enough cases (because of outbreaks and regional epidemics) still occur. In 2010, the CDC reported 27,500

cases of pertussis. This increased number of identifiable cases may be due to improved awareness and

improved diagnostic methods, such as nucleic acid-based testing.

Klebsiella spp. Rhinoscleroma is a rare form of chronic, granulomatous infection of the nasal passages,

including the sinuses and occasionally the pharynx and larynx. Associated with Klebsiella rhinoscleromatis and

Klebsiella ozaenae, the disease is characterized by nasal obstruction

appearing over a long period, caused by tumor-like growth with local extension. K. ozaenae may contribute to

another infrequent condition called ozena, characterized by a chronic, mucopurulent nasal discharge that is

often foul smelling. It is caused by secondary, low-grade anaerobic infection.

ORAL CAVITY: Stomatitis is an inflammation of the mucous membranes of the oral cavity. Herpes simplex

virus is the primary agent of this disease, in which multiple ulcerative lesions are seen on the oral mucosa.

These lesions are painful and can be found in the mouth and in the oropharynx. Herpetic infections of the oral

cavity are prevalent among immunosuppressed patients.

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Thrush: Candida spp. can also invade the oral mucosa. Immunosuppressed patients, including very young

infants, may develop oral candidiasis, called thrush. Oral thrush can extend to produce pharyngitis or

esophagitis, a common finding in patients with acquired immunodeficiency syndrome and in other

immunosuppressed patients. Thrush is suspected if whitish patches of exudate on an area of inflammation are

observed on the buccal (cheek) mucosa, tongue, or oropharynx. Oral mucositis or pharyngitis in the

granulocytopenic patient may be caused by Enterobacteriaceae, S. aureus, or Candida spp. and is

manifested by erythema, sore throat, and possibly exudates or ulceration.

Diagnosis of upper respiratory tract infections:

Collection and transport of specimens:

Sterile swabs are suitable for collecting most upper respiratory tract microorganisms. Swabs for detection of

group A streptococci (Streptococcus pyogenes) are the only exception. Throat swabs are also adequate for

recovery of adenoviruses and herpes viruses, Corynebacterium diphtheriae, Mycoplasma, Chlamydia, and

Candida spp. Recovery of C. diphtheriae is enhanced by culturing both the throat and nasopharynx.

Nasopharyngeal swabs are better suited for recovery of Bordetella pertussis, Neisseria spp., along with several

viruses including respiratory syncytial virus, parainfluenza virus, and the other viruses causing rhinitis.

nasopharyngeal secretions collected by either aspiration or washing

Direct visual examination or detection

A Gram stain of material obtained from upper respiratory secretions or lesions may not improve diagnosis.

Yeast-like cells can be identified, which are helpful in identifying thrush, and the characteristic pattern of

fusiform and spirochetes of Vincent’s angina may be visualized. Gram’s crystal violet (allowed to remain on

the slide for 1 minute before rinsing with tap water) and the Gram stain can be used to identify the spirilla and

fusiform bacilli of Vincent’s angina.

However, if crystal violet is used, the smear should be very thin because everything will be intensely Gram

positive, making a thick smear difficult to read. Additionally, spirilla and bacilli may be stained using a dilute

solution of carbol fuchsin.

For causes of pharyngitis, Gram stains are unreliable. Direct smears of exudate from membrane-like lesions

used to differentiate diphtheria from other causes are also not reliable or recommended.

Fungal elements, including yeast cells and pseudohyphae, may be visualized with a 10% potassium hydroxide

(KOH) preparation, calcofluor white fluorescent stain, or periodic acid-Schiff (PAS) stain.

Direct examination of material obtained from the nasopharynx of suspected cases of whooping cough using a

fluorescent antibody stain has been shown to yield some early positive results for detection of B. pertussis.

Various methods, including fluorescent antibody stain reagents, enzyme immunoassays, and nucleic acid

amplification methods are also commercially available to detect numerous viral agents.

 Culture of Streptococcus pyogenes (Beta-Hemolytic Group A Streptococci) are usually beta-hemolytic, with

less than 1% being nonhemolytic. Three variables must be taken into consideration regarding successful culture

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of group A streptococci from pharyngeal specimens: medium, atmosphere, and duration of incubation. Kellogg

recommended four combinations of media and atmosphere of incubation for throat specimens.

 Regardless of the medium and atmosphere of incubation employed, culture plates should be incubated for at

least 48 hours before reporting as negative for group A streptococci. In addition, the incubation of sheep blood

agar in 5% to 10% CO2 was strongly discouraged.

Drawbacks to culture include an extended incubation time of 24 to 48 hours for visible colony formation with

additional manipulations of the beta-hemolytic organisms for definitive identification. If sufficient numbers of

pure colonies are not available for identification, a subculture requiring additional incubation is necessary. By

placing a 0.04-unit differential bacitracin filter paper disk, available commercially directly

on the area of initial inoculation, presumptive identification of S. pyogenes can be made after overnight

incubation (all of group A and a very small percentage of group B streptococci are susceptible). However, use

of the bacitracin disk in the primary area of inoculation reduces the sensitivity and specificity of culture and

identification of S. pyogenes.

 Sometimes growth of too few beta-hemolytic colonies or overgrowth of other organisms makes interpretation

difficult. Therefore, using the bacitracin disk as the only method of identification of S. pyogenes is not

recommended.

 New selective agars, such as streptococcal selective agar, have been developed that suppress the growth of

almost all normal flora and beta-hemolytic streptococci except for groups A and B and Arcanobacterium

haemolyticum. Direct antigen or nucleic detection tests or the PYR test can also be carried out on isolated betahemolytic colonies.

Corynebacterium diphtheriae

If diphtheria is suspected, the physician must communicate this information to the clinical laboratory. Because

streptococcal pharyngitis is included in the differential diagnosis of diphtheria and because dual infections do

occur, cultures for Corynebacterium diphtheriae should be plated onto sheep blood agar or streptococcal

selective agar, as well as onto special media for recovery of this agent. These special media include a Loeffler’s

agar slant and a cystine-tellurite agar plate.

Recovery of this organism is improved when culturing specimens from the throat and nasopharynx of

potentially infected patients. In addition to culture, rapid toxigenicity assays, including immunoassays and

polymerase chain reaction, may be used to assist in the diagnosis. Caution should be used when interpreting

molecular assays, because positive results have been associated with related species of Corynebacteria.

Bordetella pertussis

Freshly prepared Bordet-Gengou agar was the first medium developed for isolation of Bordetella pertussis.

Today, Regan-Lowe or charcoal horse blood agar is recommended for use in diagnostic laboratories.

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Neisseria

Specimens received in the laboratory for isolation of Neisseria meningitidis (for detection of carriers) or N.

gonorrhoeae should be plated to a selective medium, either modified Thayer-Martin or Martin-Lewis agar.

After 24 to 48 hours of incubation in 5% to 10% carbon dioxide.

Epiglottitis:

Clinical specimens from cases of epiglottitis (swab sobtained by a physician) should be plated to sheep blood

agar, chocolate agar (for recovery of Haemophilus spp.), and a streptococcal selective medium. Staphylococcus

aureus, Streptococcus pneumoniae, and beta-hemolytic streptococci are all potential etiologic agents of this

disease.

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Lecture 17

Eye Diagnostic Microbiology

When we reject the specimens

1. Inappropriate specimen transport device

2. mislabeled specimen

3. Unlabeled specimen

4. specimen received after prolonged delay (usually more than two hour)

5. specimen received in expired transport media

Eye pathogens and commensals

Common pathogens commensals

Streptococcus pyogenes Staphylococcus epidermidis

Pseudomonas aeruginosa Lactobacillus spp

Chlamydia trachomatis Propionibacterium spp

Streptococcus pneumoniae Staphylococcus aureus

Haemophilus influenzae Various Enterobacteriaceae

Haemophilus aegyptius Various streptococcus spp

Specimen collection

1. Pull down the lower eyelid so that the lower conjunctival fornix is exposed.

2. Swab the fornix without touching the rim of the eyelid with the sterile cotton swab.

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3. Place the swab immediately in a bacterial transport medium or, if the specimen is brought to the laboratory immediately, in a

sterile test tube with 0.5 mL of buffered saline (pH 7)

Time relapse before processing the sample

Eye specimen should be processed immediately because tears contains lysosomes which may kill the organism

Media used in eye swab culture

A. Blood Agar plate

B. Chocolate Agar plate

C. MacConkey’s Agar plate

Result reporting

1) Report Gram stain finding as an initial report.

2) Report the isolated pathogen and its sensitivity pattern as a final report.

Turnaround time

1) Gram stain results should be available 1 hour after specimen receipt.

2) Isolation of a possible pathogen can be expected after 2-3 days.

3) Negative culture will be reported out 1-2 days after the receipt of the specimen.

Notes

a. All bacteria isolated in fair amounts and not resembling contaminants will be identified and tested for antibiotic

susceptibility, including susceptibility to chloramphenicol.

b. If trachoma is suspected, conjunctival scraping should be smeared onto a microscopic slide, air-dried and fixed in absolute

methanol. Chlamydia antigen detection systems are available for this purpose

c. Chlamydia trachomatis, an exclusively human pathogen, is a non-motile, Gram negative intracellular bacterium having a

unique developmental cycle in the infected cell. Metabolically inactive elementary body (EB) which is infectious enters the host

cell (columnar epithelial cell) and develops into a reticulate body (RB) which divides rapidly by binary fission leading to a

release of infectious

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Figure shows immunoflorescence staining showing the reticulate bodies of Chlamydia trachomatis grown on McCoy cell line

culture

Ear Diagnostic Microbiology

Etiological diagnosis of external or media otitis by aerobic and anaerobic culture with identification and susceptibility test of the

isolated organism(s).

Types of specimen

Pus from the external or middle ear.

Criteria of specimen rejection

1. Inappropriate specimen transport device

2. Mislabeled specimen

3. Unlabeled specimen

4. Specimen received after prolonged delay (usually more than two hour)

5. Specimen received in expired transport media

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Common pathogens Commensal flora at external canal

Staphylococcus aureus Staphylococcus epidermidis

Streptococcus pyogenes Lactobacillus spp.

Pseudomonas aeruginosa Propionibacterium spp.

Other Gram negative bacilli Staphylococcus aureus

Streptococcus pneumoniae Various Enterobacteriaceae

Haemophilus influenzae Various Streptococcus spp

Anaerobic bacteria Candida spp. other than albicans

Proteus spp. Occasion Pseudomonas aeruginosa

Specimen collection

1. Collect a specimen of the discharge on a thin, sterile cotton wool or Dacron swab.

2. Place the swab in a container with the transport medium, breaking off the swab stick to allow the stopper to be replaced tightly.

3. Label the specimen and send it to the laboratory.

Time relapse before processing the sample

Not more than 2 hours

Media used in eye swab culture

1) Blood Agar plate

2) Chocolate Agar plate

3) MacConkey Agar plate

Interfering factors

Patient on antibiotic therapy.

Improper sample collection.

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Result reporting

Report Gram stain finding as an initial report.

Report the isolated pathogen and its sensitivity pattern as a final report.

For external ear infections only Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, and Aspergillus will

be looked for and reported.

For middle ear infections only Pneumococcus, Streptococcus pyogenes, Haemophilus influenzae and Staphylococcus aureus will

be reported with a susceptibility test.

For the chronic discharging ear, Bacteroides species and fungi will also be reported in addition to the organisms reported for

middle ear infections.

Para nasal Sinus Diagnostic Microbiology

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Pathogens infect the sinuses

Viruses

Rhinovirus

Adenovirus

Influenzae virus

Parainfluenzae virus

Bacteria

Streptococcus pneumoniae

Haemophilus influenzae

Moraxella catarrhalis

Staphylococcus aureus

Streptococcus pyogenes

Most sinus infections are viral, and only a small proportion develops a secondary bacterial infection. Rhinoviruses, influenza

viruses, and Parainfluenzae viruses are the most common causes of sinusitis.

The most common bacteria isolated from pediatric and adult patients with community-acquired acute purulent sinusitis are

Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes. Staphylococcus aureus

and anaerobic bacteria (Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.) are the main isolates in

chronic sinusitis.

Pseudomonas aeruginosa and other aerobic and facultative gram-negative rods are commonly isolated from patients with

nosocomial sinusitis, the immunocompromised host, those with HIV infection, and in cystic fibrosis.

Fungi and Pseudomonas aeruginosa are the most common isolates in neutropenic patients. The microbiology of sinusitis is

influenced by the previous antimicrobial therapy, vaccinations, and the presence of normal flora capable of interfering with the

growth of pathogens.

The nasal cavity must disinfect with Povidone solution / chlorhexidine solution, swabs were taken from the infected sinuses. Two

swabs were taken, one for aerobic and fungus, and another for anaerobic microorganisms.

Swabs were inoculated in MacConkey agar and Chocolate agar. The specimens were incubated at 35° C in a 5% carbon dioxide

environment. The plates were evaluated daily for at least two days for any microbial growth. For anaerobic culture, swabs were

transported via thioglycolate broth and later inoculated in blood agar plates. These were incubated anaerobically at 35° C, and

evaluated for any microbial growth daily for at least five days. Fungal analysis was done by KOH mount and culture on

Sabouraud Chloroamphenicol agar plate.

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Skin (wound, abscess, burns) Diagnostic Microbiology

Types of specimen

Swabs from the infected area or aspiration from deep wounds

Swabs in anaerobic transport media for the isolation of anaerobes

Criteria of specimen rejection

1) Inappropriate specimen transport device

2) Mislabeled specimen

3) Unlabelled specimen dried samples and specimen received after prolonged delay

(Usually more than 72 hours)

4) specimen received in expired transport media

Pathogenic bacteria Commensals bacteria

Pseudomonas aeruginosa Alpha haemolytic streptococci

Proteus spp Corynebacterium spp.

E. coli Coagulase negative Staph.

Klebsiella spp Propionibacterium spp.

Morganella Bacillus spp.

Providencia

Streptococcus pyogenes

Staphylococcus aureus

Enterococcus spp.

Clostridium perfringens

Fusobactrium spp

Peptostreptococcus spp

Mycobacterium tuberculosis

Nocardia spp.

Actinomyces israelii

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Specimen collection

Pus from abscess is best to collected at the time; the abscess is incised and drained. Using sterile technique, aspirate or collect

from drainage tube up to 5ml of pus, transfer to sterile container. If pus is not being discharged use sterile cotton wool swab to

sample from the infected site, extend the swab deeply into the depth of the lesion. Immerse the swab in container of transport

medium, label it and send to the laboratory as soon as possible.

Time relapse before processing the sample

30 minutes

Storage

Maintain specimen swab at room temperature. Do not refrigerate

Media used in culture

1. Blood Agar,

2. Chocolate Agar,

3. MacConkey Agar

4. Thioglycollate broth

Culturing procedure

Streak one blood agar plates, one chocolate, MacConkey and inoculate thioglycollate broth tube. Gram stain to check the

presence or absence and if present the type or types and the predominant organisms.

Interfering factors

 Patient on antibiotic therapy

 Improper sample collection

Result reporting

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Report Gram stain finding as an initial report

Report the isolated pathogen/s and its sensitivity pattern as a final report

Turnaround time

Gram stain results should be available 1 hour after specimen receipt. Isolation of a possible pathogen can be expected after 2-3

days. Negative culture will be reported out 1-2 days after the receipt of the specimen.

Note below

Contamination of the specimen with normal flora is one of the major obstacles in obtaining good results. Care should be taken to

avoid contaminating the specimen with normal flora. This could e accomplished by swabbing superficial infected wounds with

70% alcohol before we take our swab.

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Section II - Virology By Dr. Kareem Lilo

1. General structure and classification of viruses

General properties of viruses:

1. Viruses are smaller than bacteria, they range in size between 20-300 nanometer ( nm )

(Table 2-1).

2. Viruses contain only one type of nucleic acid, either DNA or RNA, but never both.

3. Viruses consist of nucleic acid surrounded by a protein coat. Some viruses have

additional lipoprotein envelope.

4. Viruses lack cellular organelles, such as mitochondria and ribosomes.

5. Viruses are obligate cellular parasites. They replicate only inside living cells.

6. Viruses replicate through replication of their nucleic acid and synthesis of the viral

protein.

7. Viruses do not multiply in chemically defined media.

8. Viruses do not undergo binary fission.

Virus is a broad general term for any aspect of the infectious agent and includes:

• the infectious or inactivated virus particle

• viral nucleic acid and protein in the infected cell

Virion is the physical particle in the extra-cellular phase which is able to spread to new host cells;

complete intact virus particle is able to spread to new host cells; complete intact virus particle.

Table ( 2-1) : Comparison between viruses and bacteria

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The structure of viruses:

1. Viral nucleic acid:

The viral nucleic acid is located internally and can be either single- or

double- stranded RNA or DNA. The nucleic acid can be either linear

or circular. The DNA is always a single molecule, the RNA can exist

either as a single molecule or in several pieces (segmented).

• Some RNA viruses are positive polarity and others are negative polarity.

• Positive polarity is defined as an RNA with same base sequence as the mRNA. (positive

strand RNA)

• Negative polarity has a base sequence that is complementary to the mRNA. (Negative strand

RNA) ( Figure 2-1)

2. Capsid:

The protein shell, or coat, that encloses the nucleic acid genome and

mediates the attachment of the virus to specific receptors on the host

cell surface.

3. Capsomeres:

Morphologic units seen in electron microscope. Each capsomere,

consisting of one or several proteins.

Naked viruses are composed of nucleic acid + capsid (nucleocapsid)

Figure 2-1 Naked virus composition

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4. Viral envelope :

The envelope is a lipoprotein membrane composed of lipid derived from the host cell membrane

and protein that is virus- specific.

Furthermore, there are frequently glycoproteins in form of spike-like

projections on the surface, which attach to host cell receptors.

Matrix protein mediates the interaction between the capsid proteins and envelope.

The presence of an envelope confers instability on the virus Enveloped viruses NA

+ capsid + envelope

The whole virus particle is called virion. (Figure 2-2)

Types of symmetry of virus particles: (Figure 2-3)

Viruses are divided into three groups, based on the morphology of the nucleocapsid and the

arrangement of capsomeres.

1. Icosahedral symmetry

Composed of 12 vertices, has 20 faces (each an equilateral triangle) with

the approximate outline of a sphere.

e.g. Virus that cause yellow fever and Poliovirus

2. Helical symmetry

Figure 2-2 illustrate the difference between enveloped virus and naked virus

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The virus particle is elongated or pleomorphic (not spherical), and the nucleic acid is spiral.

Caposomeres are arranged round the nucleic acid.

e.g. Rabies virus

3. Complex structures

The virus particle does not confirm either cubic or helical symmetry

e.g. Poxviruses

 Reaction to physical and chemical agents:

1. Heat and cold:

Viral infectivity is generally destroyed by heating at 50-60 C0 for 30

mint., Viruses can be preserved at -90 C0 or

-196 C0 (liquid nitrogens).

2. PH:

Viruses can be preserved at physiological PH (7.3).

3. Ether susceptibility :

Ether susceptibility can be used to distinguish viruses that possess

an envelope from those that do not.

4. Detergents:

Nonionic detergents solubilize lipid constituents of viral

membranes. The viral proteins in the envelope are released.

Anionic detergents also solubilize viral envelopes; in addition, they

disrupt capsids into separated polypeptides.

5. Salts

Icosahedral Complex Helical

Figure( 2-3) Types of symmetry of virus particles

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Many viruses can be stabilized by salt in concentrations of 1 mol/L.

e.g. MgCl2, MgSO4, Na2SO4.

6. Radiation

Ultraviolet, X-ray, and high-energy particles inactivate viruses.

7. Formaldehyde

Destroys viral infectivity by reacting with nucleic acid.

8. Antibiotics

Antibacterial antibiotics have no effect on viruses.

Classification of viruses:

1. Virion morphology, including size, shape, type of symmetry,presence or absence of

envelope.

2. Virus genome properties, including type of nucleic acid (DNA or RNA), size of genome,

strandedness (single or double), whether linear or circular, positive or negative sense

(polarity), segments (number, size).

3. Physicochemical properties of the virion, including PH stability,thermal stability, and

susceptibility to physical and chemical agents,especially ether and detergents.

4. Virus protein properties, including number, size and functional activities of structural

and non-structural proteins, amino acid sequences, and special functional activities

(transcriptase, reverse transcriptase, neuraminidase, fusion activities).

5. Genome organization and replication, including gene order, strategy of replication

(patterns of transcription, translation), and cellular sites (accumulation of proteins,

virion assembly, virion release).

6. Antigenic properties

7. Biological properties, including natural host range, mode of transmission, vector

relationships, pathogenicity, tissue tropisms, and pathology.

Universal system of virus taxonomy:

Families – on the basis of virion morphology, genome structure and

strategies of replication.

Virus family names have the suffix – viridae.

Genera – based on physicochemical or serological differences.

Genus names carry the suffix – virus.

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Survey of DNA-containing

• Parvoviruses: human parvovirus B19

• Papovaviruses: papillomaviruses

• Adenoviruses: 47 types infect humans

• Herpesviruses: human herpesvirus 1-8

• Poxviruses: smallpox; vaccinia

• Hepadnaviruses: HBV

Survey of RNA-containing

• Picornaviruses

• Astroviruses

• Caliciviruses

• Reoviruses

• Arboviruses

• Togaviruses

• Flaviviruses

• Arenaviruses

• ronaviruses: SARS

• Retroviruses

• Bunyaviruses

• Othomyxoviruses

• Paramyxoviruses:

• Rhabdoviruses:rabies virus

• Bornaviruses: BDV

• Filoviruses

• Other viruses

• Viroids

Viral Replication : ( Figure 2-4)

Steps in Viral Replication:

A. Attachment:

This is the first step in viral replication. Surface proteins of the virus interact with specific

receptors on the target cell surface.

B. Penetration:

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Enveloped with the viruses (e.g., HIV, influenza virus)penetrate cells through fusion of the viral

envelope host cell membrane. Non-enveloped viruses penetrate cells by translocation of the

virion across the host cell membrane or receptor mediated endocytosis .

C. Uncoating:

This process makes the nucleic acid available for transcription to permit multiplication of the

virus.

D. Transcription and Translation:

The fact that viruses must use host cellular machinery to replicate and make functional and

structural proteins.

Assembly and Release. The process of virion assembly involves bringing together newly formed

viral nucleic acid and the structural proteins to form the nucleocapsid of the virus

E. Virus Shedding:

This is a necessary step to maintain a viral infection in populations of hosts. Shedding usually

occurs from the body surfaces involved in viral entry. Shedding occurs at different stages of

disease depending on the particular agent involved .

Figure (2-4) steps of viral replication

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Patterns of viral infection

 Viral infections can be:

- Acute (rapid and self-limiting)

- Persistent (long term)

- Latent (extreme versions of persistent infections)

- Slow or transforming (complicated types of persistent infections

Viral Pathogenesis :

Viral pathogenesis is the process by which a viral infection leads to disease.

A virus must first attach to and enter cells of one of the body surfaces: skin, respiratory tract,

gastrointestinal tract, urogenital tract, or conjunctiva.

Major exceptions are those viruses that are introduced directly into the bloodstream by needles

(hepatitis B, human immunodeficiency virus [HIV]), by blood transfusions, or by insect

vectors (arboviruses )

 Viruses usually replicate at the primary site of entry. Some, such as common cold viruses and

rotaviruses produce disease at the portal of entry and have no necessity for further systemic

spread.

Viral Spread and Cell Tropism

Many viruses produce disease at sites distant from their point of entry. Spread within the host .

Mechanisms of viral spread vary, but the most common route is via the bloodstream or

lymphatics. The presence of virus in the blood is called viremia

Routs of infection

• Inhalation ;e.g influenza viruses

• Ingestion : polio viruses

• Parentral : AIDs

• Transplacental, cytomegalovirus ,rubella.

Following infection the virus is transmitted by blood, cells, along nerves and become localized

in certain tissue which it prefer (tropism) e.g. polio, rabies etc.

Mechanisms of viral injury

1. Inhibits host cell DNA, RNA or protein synthesis e.g. Poliovirus.

2. Direct cell killing by damaging host cell membrane e.g. Rhinoviruses.

3. Induce Immune reaction e.g hypersensitivity reaction in respiratory cyncytial viruses.

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4. Damage host defiance mechanism e.g. respiratory epithelium predisposes to the

pneumonia

5. Induce cell proliferation & transformation result in neoplasia e.g. HBV, EBV.

Chemotherapy of Viral Infections :

Anti-bacterial drugs such as the penicillin antibiotics have proved very successful since they act

against a bacterial structure, the cell wall that is not present in eukaryotic cells.

In contrast, most anti-viral agents have proved of little use therapeutically since the virus uses

host-cell metabolic reactions and thus, for the most part, anti-viral agents will also be anticell agents.

A successful anti-viral drug should:

(i) interfere with a virus-specific function.

or

(ii) interfere with a cellular function so that the virus cannot replicate. To be

specific, the anti-viral drug must only kill virus-infected cells

An ideal drug should be:

• Water-soluble

• Stable in the blood stream

• Easily taken up by cells

An ideal drug should NOT be:

• Toxic

• Carcinogenic

• Allergenic

• Mutagenic

Toxicity of an anti-viral drug may be acceptable if there is no alternative: such as, for example, in

symptomatic rabies or hemorrhagic fever.

Basic Mechanisms:

Antiviral drugs specifically inhibit one or more steps of virus replication without causing

unacceptable side effects. (Figure 2-5)

Because of the close interaction between virus replication and normal cellular metabolism, it was

originally thought too difficult to interrupt the virus replicative cycle without adversely

affecting the host cell metabolism.

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The life cycle of a virus comprises several stages such as binding to the cell surface, replication,

protein synthesis etc. and all of these stages may be the target of anti-viral drugs.

The mechanism of action vary among antiviral :

1. Nucleoside and nucleotide Analogs:

The majority of available antiviral agents are nucleoside analogs. They inhibit nucleic acid by

inhibition of polymerases essential for nucleic acid replication. In addition, some analogs can

be incorporated into the nucleic acid and block further synthesis or alter its function .

Example for nucleoside analogs include acyclovir , lamivudine ,ribavirin and zidovudine : AZT

Acyclovir (Zovirax) represents a major breakthrough in the treatment of herpes virus infections.

The main indications for its use are primary genital herpes

Nucleotide analogs differ from nucleoside analogs in having an attached phosphate group, their

ability to persist in cells for long periods of time increases their potency Cidofovir is an

example .

2. Reverse transcriptase inhibitor :

It acts by binding directly to reverse transcriptase and disrupting the enzyme’s catalytic site, for

example Nevirapine.

3. Protease inhibitor :

Saquinavir was the first protease inhibitor to be approved for treatment of HIV infection , which

inhibit viral protease that is required for the last stage of replicative cycle . Inhibition of the

protease yields noninfectious virus particles.

Other types of Antivirus agent

Fuzen Blocks the virus and cellular membrane fusion step involved in entry HIV into cells.

Amantadine and Rimantadine: specifically inhibit Influenza A virus by blocking viral

uncoating.

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Interferon:

Interferon are small proteins released by macrophages, lymphocytes, and tissue cells infected

with a virus.

When a tissue cell is infected by a virus it release Interferon, Interferon will diffuse to

surrounded cells. When it binds to a receptor on the surface of these adjacent cell they begun

the production of proteins that prevents the spread of the virus through the body .

Three types of interferon: alpha, beta and gamma:

Vaccine:

A vaccine is a biological preparation that provides active acquired immunity to a

particular disease. A vaccine typically contains an agent that resembles a disease-causing

microorganism and is often made from weakened or killed forms of the microbe,

Type of viral vaccine:

1. Attenuated live viral vaccines

These attenuated viruses can infect and replicate in the recipient and produce a protective

immune response without causing disease. Live attenuated viral vaccines can often confer

(Figure 2-5) Virus replication cycle

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lifelong immunity after one immunization series (e.g. : Measles vaccine ,Rubella vaccine Oral

poliomyelitis vaccine (OPV)

2. Killed (inactivated) viral vaccines

Killed viral vaccines contain either whole virus particles, inactivated by chemical or physical

means, or some component(s) of the virus. They do not generally produce lifelong immunity

following one immunization series (e.g.: Rabies vaccine, Injectable poliomyelitis vaccine

(IPV)

3. Recombinant-produced antigens

Application of a recombinant DNA strategy to develop new vaccines. This approach has made

possible a safe and effective recombinant vaccine against hepatitis B virus, which has

replaced the vaccine derived from the plasma of hepatitis B virus-infected individuals.

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2. Virological Tests

Diagnostic Methods in Virology

1. Direct Examination

2. Indirect Examination (Virus Isolation)

3. Serology

Direct Examination

1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particles

 immune electron microscopy

3. Light Microscopy histological appearance

 inclusion bodies

4. Viral Genome Detection hybridization with specific

nucleic acid probes polymerase chain reaction (PCR)

Indirect Examination

1.Cell Culture cytopathic effect (CPE)

 haemabsorption

 immunofluorescence

2. Eggs pocks on CAM

 haemagglutination

 inclusion bodies

3. Animals disease or death

Methods for Cultivation of Virus: (Figure 2-6)

Generally three methods are employed for the virus cultivation

1. Inoculation of virus into animals

2. Inoculation of virus into embryonated eggs ( Figure 2-7)

3. Tissue culture

Figure 2-6 Method for cultivation of virus

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Laminar Flow Hoods:

 Virologist’s facility

 Laminar vertical flow hoods Contains HEPA filter Removes 99.97% of particles of 0.3μM

or higher

Figure2-8 laminar vertical flow hoadis

Figure 2-7 Inoculation of virus into embryonated eggs

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Common Methods:

Quantitative assays

• Plaque assays

• TCID50

• Haemagglutination assays

• Transformation assays

Virus Isolation :

Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:

1. Primary cells - Monkey Kidney

2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts

3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK

Primary cell culture are widely acknowledged as the best cell culture systems available since

they support the widest range of viruses. However, they are very expensive and it is often

difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the

range of viruses supported is often limited.

Cell culture Cytopathic Effect :

• Some viruses kill the cells in which they replicate, and infected cells may eventually

detach from the cell culture plate.

• As more cells are infected, the changes become visible and are called cytopathic effects.(

Figure 2-9)

Figure 2-9 cell culture cytopathic effects