Viral dissemination in the organism

There are two forms of infection:

i. Local infection
In this form of infection, the viruses spread only from cell to cell.
The infection and manifest disease are thus restricted to the tissues in the immediate vicinity of
the portal of entry. Example: rhinoviruses that reproduce only in the cells of the upper respiratory
tract.

ii. Generalized infection

 In this type, the viruses usually replicate to some extent at the portal of entry and are then
disseminated via;
a. The lymph ducts or bloodstream and reach their target organ either directly or after infecting a
further organ.
 When the target organ is reached, viral replication and the resulting cell destruction become
 so widespread that clinical symptoms develop.
b. Neurogenic spread along the nerve tracts, from the portal of entry to the CNS (rabies), or
in the opposite direction from the ganglions where the viruses persist in a latent state to the
target organ (herpes simplex).
 TOPIC FIVE
ANTIVIRAL CHEMOTHERAPY
 Many viruses encode activities (virulence factors) that promote the efficiency of viral
replication, viral transmission, the access and binding of the virus to target tissue, or escape
of the virus from host defense and immune resolution.
 Loss of virulence factors results in attenuation of the virus.
 Viruses also encode enzymes that are not present in un-infected cell.
 These enzymes are critical to viral replication but unnecessary for cellular function.
 It is now possible to find specific inhibitors for viral growth.
Mechanisms of antiviral chemotherapy
Antiviral chemicals work in the following ways:
i. Inhibitors of nucleic acid synthesis.
ii. The stages of attachment of virus to host cell
iii. Stage of uncoating of the viral genome
iv. Reverse transcription of certain viral genomes
v. Regulation of viral transcription
vi. Replication of viral nucleic acid
vii. Translation of viral proteins
viii. Effects on assembly of viral parts
ix. Effects on viral maturation
x. Release of progeny virus
→ The mechanism of action varies among anti-virals.
Antiviral agents and their targets
 Viral vaccines
 Purpose of viral vaccines is to utilize immune response of the host to prevent viral disease
 Several remarkably success include;
i. Eradicated of small pox
ii. Reducing annual incidence of several viral diseases
Working of Vaccines
i. Stimulates adaptive immune response
 Natural infection prevents reoccurrence of disease
 Humoral & Cellular responses
ii. Vaccines prevent or modify disease. Most do NOT prevent infection.
iii. Herd immunity reduces spread of disease.
 Disease agents require a certain level of transmission to be maintained
Requirements of an effective Vaccine
 To be effective a vaccine should be capable of eliciting the following;
i. Activation of Antigen-Presenting Cells to initiate antigen processing and producing
interleukins.
ii. Activation of both T and B cells to give a high a high yield of memory cells.
iii. Generation of Th and Tc cells to several epitopes, to overcome the variation in the immune
response in the population due to MHC polymorphism.
iv. Persistence of antigen, probably on dendritic follicular cells in lymphoid tissue, where B
memory cells are recruited to form antibody-secreting cells that will continue to produce
antibody.
Types of viral Vaccines
 Vaccine types can broadly be classified into three groups:
1. Whole-organism Vaccines
A. Inactivated (Killed) Vaccine
B. Live-attenuated vaccines
C. Chimeric vaccine
2. Subunit Vaccines
A. Polysaccharide Vaccine
B. Conjugated Vaccines
 C. Toxoid Vaccines
D. Recombinant Protein Vaccines
E. Nanoparticle vaccines
3. Nucleic Acid Vaccines
A. DNA plasmid vaccines
B. mRNA vaccines
C. Recombinant vector vaccine

1. Whole organism vaccines

 Made of an entire pathogen
 We have two major categories of whole organism vaccines
A. killed (inactivated) or
B. weakened (attenuated)
A. Inactivated (Killed) Vaccine
 These were produced by killing the pathogen (bacteria, virus, or other pathogens) with
chemicals or heat, or radiation.
 The killed pathogen cannot cause disease, and this means that they do not replicate in the
host’s body.
Advantage: These vaccines are stable and safer than the live attenuated vaccines
Disadvantage: The vaccine elicits a weaker immune response and therefore, it requires more
vaccine dosages and a booster dose as well, so as to confer protective immunity.
Examples of Inactivated Vaccines
i. Poliomyelitis (sulk vaccine),
ii. Rabies,
iii. Typhoid,
iv. Cholera,
v. Pertussis,
vi. Pneumococcal,
vii. Rabies,
viii. Hepatitis B
ix. Influenza vaccines.
B. Live-attenuated vaccines
 These vaccines are prepared from a whole organism, by weakening their pathogenicity.
 The organism cannot cause disease but can induce an immune response, hence the
term attenuation.
 These vaccines elicit strong immune responses because they are similar to the actual
disease pathogen and hence they confer a life-long immunity
Examples
i. Measles/Mumps/Rubella (MMR) and
ii. Influenza Vaccine
iii. Polio (Sabin vaccine),
iv. Rotavirus,
v. Tuberculosis,
vi. Varicella,
vii. Yellow fever.
 The attenuated strain of Mycobacterium bovis called Bacillus Calmette- Guérin (BCG) was
developed by growing M. bovis on a medium containing increasing concentrations of bile.
 After 13 years, this strain had adapted to growth in strong bile and had become sufficiently
attenuated that it was suitable as a vaccine for tuberculosis.
C. Chimeric vaccine
 The vaccines contain genetic information from one viral particle that display the biological
properties of different parent viruses
Example
 An NIAID-developed live-attenuated chimeric vaccine consisting of a dengue virus
backbone with Zika virus surface proteins is undergoing early-stage testing in humans.
2. Subunit vaccines
 These are vaccines that are prepared by using components or antigens of the pathogen.
 These components can stimulate the immune system to elicit appropriate immune
responses.
 Subunit vaccines include:
a. Polysaccharide Vaccine
  They are vaccines that are prepared using the sugar molecules, and polysaccharides from
the outer layer of a bacteria or virus.
Examples of polysaccharide vaccines
i. Meningococcal disease vaccine
ii. Pneumococcal disease vaccine.
b. Conjugated Vaccines
 These vaccines are prepared by linking the polysaccharides or sugar molecules on the outer
layer of the bacteria to a carrier protein antigen or toxoid from the same microbe.
Example
i. Haemophilus influenzae type B vaccine
ii. Pneumococcal vaccine
iii. Meningococcal infections.
c. Toxoid Vaccines
 Are prepared from inactivated toxins, by treating the toxins with formalin, a solution of
formaldehyde, and sterilized water.
 This process of inactivation of toxins is known as detoxification and the resultant inactive
toxin is known as a toxoid.
Examples of toxoid vaccines
i. Diphtheria
ii. Tetanus toxoid vaccines.
d. Recombinant Protein Vaccines
 Recombination involves combining from two or more sources.
 This technology harnessed the development of recombinant protein vaccines.
Example- Hepatitis B vaccines.
e. Nanoparticle vaccines
 Nanoparticles are used to present the vaccine inform of protein subunit antigens into the
immune system.
3. Nucleic acid vaccines
 These are vaccines that introduce the genetic materials that code the antigen or the antigen
that is aimed at inducing an immune response, enabling the host cells to use the genetic
materials to produce the antigens.
Advantages of the nucleic acid vaccine
i. Stimulating a broad long-term immune response
ii. Excellent vaccine stability
iii. Ease of large-scale vaccine manufacture
iv. Rapid production
v. Reduces potential risks of working with the live pathogen
vi. Encoding only the key antigen without including other proteins
Categories of nucleic acid vaccines
a. DNA plasmid vaccines
 These are vaccines that are composed of a small circular piece of DNA known as a plasmid.
 The plasmid carries genes that encode proteins from the pathogen of interest.
Examples of DNA plasmid vaccines
i. SARS coronavirus (SARS-CoV) vaccine
ii. H5N1 avian influenza vaccine
iii. H1N1 pandemic influenza vaccine
iv. Zika virus vaccine.
b. mRNA vaccines
Prepared from mRNA which is an intermediary between DNA and protein.
Example
Zika virus vaccines that protect monkeys against Zika virus infection.
3. Recombinant vector vaccine
 These are vaccines designed as vectors or carriers using harmless viruses or bacterium and
they introduce the genetic material into cells.
Examples
HIV vaccine,
Zika virus vaccine,
Ebola virus vaccine.
 Live vs Dead vaccines

Side effects of vaccines

 The most common side effects after vaccination are mild. They include:
i. Pain, swelling, or redness where the shot was given
ii. Mild fever
iii. Chills
iv. Feeling tired
v. Headache
vi. Muscle and joint aches
vii. Fainting
NB-most common side effects are a sign that your body is starting to build immunity (protection)
against a disease.
Serious side effects
 Serious side effects from vaccines are extremely rare.
 For example, if 1 million doses of a vaccine are given, 1 to 2 people may have a severe
allergic reaction.
 Severe allergic reaction can include:
i. Difficulty breathing
ii. Swelling of your face and throat
iii. A fast heartbeat
iv. A bad rash all over your body
v. Dizziness and weakness
 TOPIC SIX
DIAGNOSIS OF VIRAL DISEASES
Stages of viral disease diagnosis
i. Clinic: presumptive diagnosis based on clinical signs and history
ii. Pathology: Observed lesions and pathological changes at gross and histopathological
levels. History may provide clues.
iii. Microbiological diagnosis: confirmatory diagnosis
Principles of microbiological diagnosis
i. Isolation of viruses
ii. Detection of viral nucleic acid/specific genes
iii. Detection of viral antigen
iv. Detection of specific virus-induced antibody
Sample collection
a. Samples must be collected from the right site and at the right time
b. Samples to be collected must relate to the clinical signs and pathological changes observed
c. Samples must be collected as soon as clinical signs are observed
d. Knowledge of the pathogenesis of the disease may dictate the type of sample to be collected
e. Proper labeling of samples for identification and to avoid confusion
f. Samples must be sent to the laboratory with history and tentative diagnosis
g. Transport samples with ice packs (4 oC) if transit time is less than 24 hours
h. For transit above 24 hours, use dry ice at -70 OC.
i. Long term storage is achieved with liquid nitrogen at -196 OC.
j. Use transport medium containing buffer (isotonic saline) with bovine albumin/foetal calf
serum (protein to prolong virus survival), antibiotic and antifungal agent (to prevent contaminants)
Samples for diagnosis of viral diseases:
i. Respiratory tract infection- nasal swab, tracheal swab, nasopharyngeal aspirate, lung tissue.
ii. Enteric infection- Faeces, rectal swab.
iii. Genital tract infection- prepucial washing, semen, genital swab.
iv. Eye infection- Conjunctival swab.
v. Skin infection- Vesicular fluid, epithelial scrapings, biopsy of solid lesions.
vi. Central nervous system- cerebrospinal fluid, faeces, nasal swab, brain tissue.
vii. Generalized infection- Nasal swab, faeces, blood leukocytes.
viii. Post mortem examination- relevant organ.
ix. Every case- Blood for serum to be used in serology.
Virus isolation:
Advantages:
i. It allows for further studies on the virus isolate
ii. It is required for preparation of vaccines
iii. It is required for preparation of antigens for rapid diagnostic kits
iv. It is highly sensitive and reliable
Disadvantages:
i. It is slow and time consuming
ii. It is labour intensive
iii. It is expensive to acquire and maintain required facilities
iv. Some viruses may not grow
v. Selection of appropriate media may require critical consideration
Methods of virus isolation
A. Virus isolation in cell culture
B. Virus isolation in embryonated egg
C. Virus isolation in laboratory animals
D. Virus isolation in susceptible host
E. Virus isolation in arthropods

A. Virus isolation in cell culture

 This is the most commonly used method of virus isolation.
Procedure
i. Inoculation of sample onto confluent tissue culture monolayer.
ii. incubation at 35-37 OC.
 The culture is examined daily for evidence of virus growth (cytopathic effect, interferon,
haemadsorption and antigen detection).
iii. Harvesting of the virus from the culture by freeze-thawing and low-speed centrifugation
iv. Use of supernatant as antigen for virus identification
 v. Detection the effect of the growing virus in cell culture by light microscopy
Types of tissue culture
i. Primary tissue culture:
 This is made directly from tissue.
 It may involve one or two passage.
 In primary tissue culture, there are mixed cell types.
 It is good for isolation of influenza virus, parainfluenza virus and enteroviruses.
Types of Primary tissue culture
 Three types are recognized:
a. Monolayer:
 Derived from tissue taken directly from the susceptible host (monkey kidney, mouse
embryo).
 The tissue is cut into very small pieces and digested into cells by proteolytic enzymes such
as trypsin or collagenase.
 The cells are grown on glass or plastic surface as monolayer in as artificial medium
containing growth factors supplemented with foetal calf serum and antibiotics.
Type of artificial medium
i. Growth medium:
 Used for cell cultivation.
 Its constituents include salts at physiological concentration, glucose, amino acids, vitamins,
antibiotics and antifungal agents and 10-20% foetal calf serum.
 It is maintained at pH 7.2-7.4.
 After formation of confluent monolayer, the growth medium is changed to maintenance
medium.
ii. Maintenance medium:
 Contains similar constituents as growth medium except that the foetal calf serum is 2 -5%
for survival of cells and no further division.
b. Suspension culture:
 Cells are grown in form of suspensions in growth medium and not monolayer.
 Lymphocyte cultures are cultivated in suspension.
c. Organ culture: organ culture are not trypsinized into cell but grown as whole. Examples
include tracheal ring organ culture.
ii. Secondary culture:
 They are obtained from primary culture by passages or subcultivation.
 Repeated passages of primary culture will produce cell lines with almost homogenous cell
type.
Types of secondary culture
a. Semi continuous cell line:
 This is derived from fibroblastic cell of animal or human foetal tissue.
 They have been subcultured through about 50 passages (generations of repeated
subcultivation).
 They have diploid number of chromosome.
 They are used for vaccine production.
 They have limited life span. Examples include HDCS, MRC-9, WI-38.
 This type of cell culture is good for herpes simplex and rhinovirus.
b. Continuous cell line:
 This is also called established cell line.
 They are widely used fro diagnostic purposes.
 They are derived from neoplastic cells or from normal cells that have been transformed by
repeated subcultivation to behave like tumour cells.
 Continuous cell line can grow indefinitely.
 They have heteroploid (aneuploid) chromosome (variable/abnormal number of
chromosome).
 It is good for vaccine production and research purposes.
Examples
i. Mardin Darby bovine kidney (MDBK),
ii. MDCK,
iii. Vero cell (African green monkey kidney cells),
iv. Hep,
v. HeLa,
vi. Crandell feline kidney (CRFK).
Detection of viral growth in cell culture
1. Observation for cytopathic effect:
 evidence of growth of virus in cell culture is by the detection of cytopathic effect (CPE).
 Cytopathic effect is defined as degenerative changes caused by the growth of viruses in
cell culture or virus-induced damage in cell culture.
Examples of cytopathic effect (observed by microscopy)
i. Cell lysis or cell disintegration
ii. Syncythial formation: formation of multinucleated giant cell due to cell fusion. This is
found with lentivirus, herpesviruses, paramyxoviruses
iii. Rounding up of cell or cellular transformation (change from spindle shape to spherical
shape)
iv. Formation of intranuclear (adenovirus, herpesvirus, parvovirus) or intracytoplasmic
(poxvirus, rhabdovirus, reovirus) inclusion bodies.
 Viruses can be categorized into burster or creeper viruses based on the type of CPE
produced.
a. Burster (lytic) viruses: these induce cell lysis and cellular transformation in cell culture
b. Creeper viruses: these induce formation of multinucleated giant cells.
1. Plaques:
 Macroscopic/gross observation of CPE on monolayer cell by overlaying with molten
agar.
 Plaques are foci of dead virus-infected cells which do not take up the stain (acridine
dye) when stained and so appear as clear spots in a stained monolayer.
2. Haemadsorption:
 Adherence of erythrocytes to monolayer cells because of the growth of
haemagglutinating virus in the cells.
 Haemagglutinin is expressed on the cell membrane of cells in monolayer and this
attracts and binds erythrocytes.
 Feline panleukopaenia virus haemagglutinate porcine erythrocyte while porcine
parvovirus haemagglutinate chick, guinea pig, monkey, human and cat erythrocytes.
3. Immunofluorescence
4. Interference
 5. Virus neutralization test
6. Haemagglutination inhibition test

B. Virus isolation in embryonated egg

 This is no longer widely used
 However, it remains the most preferred for isolation of influenza A virus and avian viruses
 10 to 12 day old embryonated eggs are used
 Eggs must be from specific pathogen-free flocks
Route of administration into embryonated egg:
This is determined by tissue affinity of the particular virus and includes;
i. Allantoic cavity
ii. Amniotic cavity
iii. Yolk sac
iv. Chorioallantoic membrane (CAM)
v. Intravascular inoculation in well-developed chick embryo
Evidence of virus growth in embryonated egg:
i. Death of the embryo
ii. Dwarfing/stunting of embryo
iii. Formation of pock on the CAM
iv. Virus isolation in laboratory animals
C. Virus isolation in laboratory animals
 Suckling mice is used for cultivation of arthropod-borne viruses.
 Suckling mice is inoculated intracerebrally and observed for signs of disease and death.
 Virus is identified from specimen collected from infected animal by complement fixation
test, virus neutralization test or by other rapid diagnostic method.
D. Virus isolation in susceptible host
 This involves inoculation of natural host species of the virus.
 This is used for evaluation of vaccine, production of polyclonal antibody and for
pathogenicity testing/verification of Koch’s postulate.
E. Virus isolation in arthropods
This is not a common method and is obsolete.
Example includes isolation of Dengue virus in adult Toxorhynchites, male Aedes aegypti and
Aedes albopictus mosquitoes by intrathoracic inoculation.

RAPID DIAGNOSTIC TECHNIQUES

These include:
1. Electron microscopy
2. Viral nucleic acid detection
3. Serology/detection of viral antigen and antibody
1. Electron microscopy
Electron microscopy is Used;
i. To demonstrate the presence of virus in clinical specimens
ii. To study the morphology or symmetry of the virus.
iii. To recognize mixed viral infection.
iv. To detect non-viable viruses and
v. To identify viruses that cannot be grown in vitro.
Drawbacks of electron microscopy
i. However, equipment required for the procedure is rather expensive.
ii. Besides, large number of viral particles in excess of 10 /ml must be present in the sample
before they can be detected.
iii. It is difficult to differentiate viruses with similar morphology especially those from the
same family with electron microscopy.
2. Detection of viral nucleic acid:
 Oligonucleotide probes are used for the detection of viral DNA or RNA.
 Insufficient viral nucleic acid in sample is increased by amplification using polymerase
chain reaction.
3. Serology
For the detection of viral antigen or virus-induced antibody in the serum of the host

Interference
 Interference occurs when infection of a host cell with one virus prevents superinfection of
the same cell with another virus.
  This is a situation that plays out when the multiplication of a particular virus in a host cell
population leads to resistance against superinfection and multiplication of another virus in
the same host cell population.

Types of Interference
i. Heterologous interference- Interference between two completely different viruses.
ii. Homologous interference- Interference between two related viruses or two strains of
viruses.
iii. Autointerference- Interference between a virus and its defective particles.
Mechanism of Interference
i. Change on receptor sites thereby preventing the attachment of the second virus
ii. Competition for cellular sites and enzymes
iii. Altering the metabolic pathway for the replication of the second virus
iv. Inducing the production of interferon
Interferons
 Interferons are non-viral proteins with molecular weight of about 20 Kilo Dalton (KDa)
produced by cells (especially leukocytes and fibroblasts) in response to virus infection and
stimulation by natural or synthetic double stranded RNA and Chlamydiae.
Classes of Interferons
i. Type I interferons
 These are alpha and beta interferons (IFN-α and IFN-β). Alpha IFN is sunthesized by virus-
infected monocytes, macrophages, epithelial cells and fibroblasts while
 IFN-β is produced by epithelial cells and fibroblasts.
ii. Type II interferon:
 Are gamma interferon (IFN-).
 They are produced by activated T-cells in response to previously encountered antigen.
Properties of Interferons
i. They are produced very early in the course of virus infection (usually within 48 hours)
ii. They are not produced by virus but by virus-infected cells
iii. They are not virus specific: Interferon produced against a virus is equally effective against
another virus
 iv. They are species specific: Interferon produced in one species of animals is not effective in
another species
v. They do not directly kill viruses
vi. They do not act as antibody
vii. Double stranded RNA are the principal inducers of interferons
Major functions of Interferons
i. They inhibit viral replication
ii. They inhibit cell division
iii. They modulate immune response to infection
iv. They enhance the display of histocompatibility antigens on cell surfaces which helps in
antigen-driven activation of T-cells
v. They modulate B- and T- cells activities
vi. They enhance the cytotoxicity of natural killer (NK) and cytotoxic T cell (TC)