VIROLOGY AND TISSUE CULTURE

INTRODUCTION TO VIRUSES

Definition and Nature of Viruses

Viruses are submicroscopic infectious agents that can infect all types of life forms, from
animals and plants to microorganisms, including bacteria (bacteriophages) and archaea. Unlike
living organisms, viruses lack cellular structure and cannot carry out metabolic processes on
their own. They rely entirely on the host cell's machinery for replication and propagation.

Historical Background

The concept of viruses began to form in the late 19th century when scientists discovered that
certain infectious diseases could be caused by agents smaller than bacteria. The term "virus"
comes from the Latin word for poison. The Key milestones include:

- 1892: Dmitri Ivanovsky's discovery of the tobacco mosaic virus.

- 1915: Discovery of bacteriophages by Frederick Twort and Félix d'Hérelle.

- 1930s: Development of the electron microscope allowed visualization of viral particle.

Structure of Viruses

Viruses consist of genetic material, either DNA or RNA, enclosed in a protein coat called a
capsid. Some viruses also have a lipid envelope derived from the host cell membrane.

- Capsid: is the protein shell that encloses the viral genome. It is composed of protein subunits
called capsomeres.

- Envelope: Some viruses have an outer lipid envelope with embedded glycoproteins, which
assist in host cell recognition and entry.

- Genome: Viral genomes can be either DNA or RNA, single-stranded or double-stranded, and
vary greatly in size and complexity.

Classification of Viruses

(I) International Committee on Taxonomy of Viruses (ICTV): Classifies viruses based on

morphology, genome structure, replication strategy, and host range.

(II) Baltimore Classification: Based on the type of nucleic acid (DNA or RNA) and replication
strategy, categorizing viruses into seven groups:

1. Group I: Double-stranded DNA viruses (e.g., Herpesviruses)

2. Group II: Single-stranded DNA viruses (e.g., Parvoviruses)

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 3. Group III: Double-stranded RNA viruses (e.g., Reoviruses)

4. Group IV: Positive-sense single-stranded RNA viruses (e.g., Picornaviruses)

5. Group V: Negative-sense single-stranded RNA viruses (e.g., Orthomyxoviruses)

6. Group VI: RNA reverse-transcribing viruses (e.g., Retroviruses like HIV)

7. Group VII: DNA reverse-transcribing viruses (e.g., Hepadnaviruses like HBV)

Viral Genetics and Replication

DNA Viruses: Can be single-stranded (ssDNA) or double-stranded (dsDNA). Examples include

Herpesviruses (dsDNA), Parvoviruses (ssDNA).

RNA Viruses: Can be single-stranded (ssRNA) or double-stranded (dsRNA). ssRNA viruses can
be positive-sense (+ssRNA) or negative-sense (-ssRNA). Examples: Coronaviruses (+ssRNA),
Influenza viruses (-ssRNA), Rotaviruses (dsRNA). Retroviruses: Have +ssRNA genomes but
replicate through a DNA intermediate e.g. HIV.

Viral Replication Cycle

1. Attachment: The virus binds to specific receptors on the surface of the host cell.

2. Penetration: The viral genome enters the host cell. This can occur via fusion with the cell
membrane or endocytosis.

3. Uncoating: The viral capsid is removed, releasing the viral genome into the host cell's
cytoplasm.

4. Replication and Transcription: The host cell's machinery is hijacked to replicate the viral
genome and synthesize viral proteins.

5. Assembly: New viral particles are assembled from the replicated genome and synthesized
proteins.

6. Release: New virions are released from the host cell either by lysis (breaking the cell open) or
budding off from the cell membrane.

Viral Pathogenesis and Epidemiology

Viral pathogenesis refers to the mechanisms by which viruses cause disease in their hosts,
while epidemiology focuses on the patterns, causes, and effects of health and disease
conditions in populations.

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Mechanisms of Viral Pathogenesis:

- Entry and Initial Replication: Viruses enter the host through various routes (respiratory tract,
gastrointestinal tract, skin, mucous membranes, blood). The initial replication occurs at the site
of entry before systemic spread.

- Cellular Tropism: this refers to the specificity of a virus for particular host cells, determined by
receptor availability and intracellular factors. Example; HIV targets CD4+ T cells, Influenza
targets respiratory epithelial cells.

- Spread and Dissemination: Localized infections remain at the entry site (e.g., rhinovirus in the
upper respiratory tract), whereas, systemic infections spread through the bloodstream (viremia)
or the nervous system (neurotropic viruses).

Cell Damage and Host Responses

- Direct Cytopathic Effects (CPE): Virus-induced cell death through mechanisms like apoptosis,
necrosis, or lysis.

- Immune-Mediated Damage: Immunopathology caused by the host's immune response, such

as cytokine storms or immune complex deposition.

- Oncogenesis: Some viruses induce cell transformation and cancer (e.g., HPV causing cervical
cancer, EBV associated with Burkitt's lymphoma).

Stages of Viral Disease:

1. Incubation Period: Time between initial infection and onset of symptoms. The Virus replicates
but no symptoms are evident yet.

2. Prodromal Period: Early, non-specific symptoms appear (e.g., fever, malaise).

3. Acute Phase: Characteristic symptoms of the disease manifest. Peak of viral replication and
immune response.

4. Convalescence: Symptoms resolve as the immune system clears the virus. It is a recovery
phase where the host regains health.

5. Chronic and Latent Infections: The chronic infection is a persistent infection with continuous
virus production (e.g., hepatitis B). In latent infection, the virus remains dormant with potential
reactivation (e.g., herpesviruses).

Host Factors Influencing Pathogenesis:

- Genetic Factors: Host genetics can influence susceptibility and disease severity (e.g., CCR5-
Δ32 mutation providing resistance to HIV).

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- Age and Immune Status: Infants, elderly, and immunocompromised individuals are at higher
risk for severe infections.

- Co-infections and Comorbidities: Concurrent infections and underlying health conditions can
exacerbate disease.

Viral Epidemiology

- Incidence: The number of new cases of a disease in a specified period.

- Prevalence: The total number of cases of a disease at a given time.

- Endemic: Constant presence of a disease within a geographic area (e.g., dengue in tropical
regions).

- Epidemic: A sudden increase in the number of cases above what is normally expected (e.g.,
Ebola outbreaks).

- Pandemic: An epidemic that spreads across multiple countries or continents (e.g., COVID-19).

Transmission

- Direct Transmission: Person-to-person spread through contact, droplets, or bodily fluids (e.g.,
influenza, HIV).

- Indirect Transmission: Via contaminated surfaces (fomites), vectors (e.g., mosquitoes for Zika
virus), or environmental sources.

- Zoonotic Transmission: Viruses transmitted from animals to humans (e.g., coronaviruses,

hantaviruses).

Factors Influencing Viral Spread

- Host Factors: Immunity, population density, behavior (e.g., travel, social interactions).

- Viral Factors: Infectivity, virulence, environmental stability.

- Environmental Factors: Climate, seasonality, vector presence.

Control and Prevention Measures:

- Vaccination: Immunization to build population immunity and prevent outbreaks (e.g., measles,
polio).

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- Quarantine and Isolation: Separating infected individuals to prevent spread.

- Public Health Interventions: Hygiene practices, vector control, and education campaigns.

- Surveillance: Monitoring and tracking disease spread to inform public health actions.

HERPES VIRUSES

Herpes viruses are a family of viruses known as Herpesviridae. This family includes several well
-known viruses that cause various diseases in humans. The most common herpes viruses
include:

1. Herpes Simplex Virus (HSV): HSV-1 causes oral herpes, leading to cold sores or fever blisters
around the mouth. HSV-2 Mainly causes genital herpes, resulting in sores and blisters in the
genital and rectal areas.

Transmission

HSV-1 is primarily transmitted through oral contact, such as kissing or sharing utensils, leading
to oral herpes. It can also be transmitted through oral-genital contact, causing genital herpes.
HSV-2 is mainly transmitted through sexual contact, including vaginal, anal, and oral sex, leading
to genital herpes.

Treatment

Antiviral medications: Drugs like acyclovir, valacyclovir, and famciclovir can reduce the severity
and duration of outbreaks and help prevent recurrence.

Topical treatments: Creams or ointments containing antivirals can be applied directly to sores
to alleviate symptoms.

Pain relief: Over-the-counter pain relievers like ibuprofen or acetaminophen, and topical
anesthetics can help manage pain and discomfort.

2. Varicella-Zoster Virus (VZV): Causes chickenpox (varicella) in children and can reactivate
later in life to cause shingles (herpes zoster).

Transmission

Chickenpox is tansmitted through respiratory droplets when an infected person coughs or

sneezes, or by direct contact with the fluid from chickenpox blisters. Shingles refers to the
reactivation of the virus in someone who previously had chickenpox. It can spread through
direct contact with the fluid from shingles blisters, but only to someone who has not had
chickenpox or the chickenpox vaccine, causing chickenpox, not shingles.

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Treatment

Chickenpox: Usually self-limiting in children, treatment focuses on symptom relief, such as

antihistamines for itching, and acetaminophen for fever.

Shingles: Antiviral medications (acyclovir, valacyclovir, famciclovir) can reduce the severity and
duration of shingles. Pain relief can include analgesics, corticosteroids, and nerve pain
medications like gabapentin or pregabalin.

3. Epstein-Barr Virus (EBV): causes infectious mononucleosis, also known as mono or the
"kissing disease." It is also associated with certain types of cancer, such as Burkitt's lymphoma
and nasopharyngeal carcinoma. Symptoms may include fatigue, fever, sore throat, enlargement
of the spleen, headache, muscle weakness, loss of appetite, swollen lymph nodes in the neck
and armpits etc.

Transmission

Primarily transmitted through saliva, often through kissing, sharing drinks, or eating utensils. It
can also be spread through blood and semen during sexual contact, blood transfusions, and
organ transplantation.

Treatment

Infectious mononucleosis: There is no specific antiviral treatment. Management includes rest,

hydration, pain relievers (ibuprofen, acetaminophen), and corticosteroids in severe cases

4. Cytomegalovirus (CMV): Typically causes mild symptoms but can be severe in

immunocompromised individuals and newborns. It can lead to serious conditions such as
retinitis, pneumonia, and hepatitis. Symptoms may include fatigue, fever, swollen lymph nodes,
sore throat, headache, abdominal pain, loss of appetite, nausea and vomiting etc

Transmission

Spread through bodily fluids, including saliva, urine, blood, breast milk, semen, and vaginal fluids.
It can be transmitted through close contact with infected individuals, sexual contact,
breastfeeding, organ transplants, and blood transfusions.

Treatment

Antiviral medications such as ganciclovir, valganciclovir, foscarnet, and cidofovir are used,
particularly in immunocompromised patients. CMV immune globulin may also be used for
prevention in certain high-risk patients.

5. Human Herpesvirus 6 (HHV-6): causes roseola (sixth disease) in infants and young children.
It is a highly contagious viral infection and is characterized by high fever, rash and swelling of
the lymph nodes, loss of appetite It can also cause complications in immunocompromised

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patients.

Transmission

Transmitted primarily through respiratory droplets from coughing or sneezing, as well as

through close personal contact.

Treatment

Roseola: Usually (self-limiting and most children recoverwithin 7-10 days) with treatment
focused on managing fever and symptoms with antipyretics and fluids to prevent dehydration.
Severe cases in immunocompromised individuals may require antiviral treatment.

6. Human Herpesvirus 7 (HHV-7): similar to HHV-6 and often co-infects with it, causing similar
symptoms.

Transmission

Spread through saliva, as well as sexual contact, blood transfusions, and organ transplants. It is
more common in individuals with weakened immune systems, such as those with HIV/AIDS.

7. Kaposi's Sarcoma-Associated Herpesvirus (KSHV or HHV-8): is associated with Kaposi's

sarcoma, a type of cancer that often affects individuals with weakened immune systems, such
as those with AIDS.

Herpes viruses are characterized by their ability to establish lifelong infections in their hosts.
They can remain latent (dormant) within the body and reactivate under certain conditions, such
as stress, illness, or immunosuppression.

IMMUNE RESPONSE TO VIRAL INFECTION

The immune system employs a multi-faceted defense strategy to combat viral infections. This
response can be broadly divided into innate and adaptive immunity, each playing a vital role in
identifying and eliminating viral invaders.

1. Innate Immune Response

The innate immune system provides the first line of defense against viral infections, offering a
rapid but non-specific response. The innate immune response include:

- Physical and Chemical Barriers: Skin, mucous membranes, and secretions such as saliva and
stomach acid act as initial barriers.

- Pattern Recognition Receptors (PRRs): Cells of the innate immune system, such as
macrophages and dendritic cells, use PRRs like Toll-like receptors (TLRs) to detect viral

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components (e.g., viral RNA or DNA).

- Interferons (IFNs): Upon detecting a virus, infected cells release type I interferons (IFN-α and
IFN-β) that induce antiviral states in neighboring cells and enhance the activity of natural killer
(NK) cells and dendritic cells.

- Natural Killer (NK) Cells: NK cells recognize and kill virus-infected cells by detecting changes in
the expression of certain cell surface molecules.

- Complement System: A group of proteins that can be activated directly by pathogens or

indirectly by pathogen-bound antibodies, leading to the lysis of infected cells and the promotion
of inflammation.

Mechanisms of the innate immune response

- Viral Recognition: PRRs identify viral components, triggering signaling pathways that activate
transcription factors such as NF-κB and IRFs (Interferon Regulatory Factors), leading to the
production of interferons and pro-inflammatory cytokines.

- Cytokine and Chemokine Release: These signaling molecules attract immune cells to the site
of infection and enhance their antiviral activities.

- Phagocytosis: Macrophages and neutrophils engulf and digest viral particles and infected cells.

2. Adaptive Immune Response

The adaptive immune response is slower to activate but provides a highly specific and long-
lasting defense against viruses. The components of the adaptive immune response include:

- B Cells: Produce antibodies that neutralize viruses and mark them for destruction.

- T Cells: Include helper T cells (CD4+ T cells) that aid other immune cells, and cytotoxic T
lymphocytes (CTLs or CD8+ T cells) that directly kill infected cells.

- Memory Cells: Both B and T cells can form memory cells that provide a quicker and stronger
response upon subsequent exposures to the same virus.

Mechanisms of the adaptive immune response

- Antigen Presentation: Dendritic cells and macrophages present viral antigens on their surface
via Major Histocompatibility Complex (MHC) molecules to T cells.

- Helper T Cell Activation: Helper T cells recognize antigens presented by MHC class II
molecules and release cytokines that activate B cells and cytotoxic T cells.

- B Cell Activation and Antibody Production: B cells that encounter their specific antigen (often
with help from helper T cells) differentiate into plasma cells that secrete antibodies. These

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antibodies can neutralize viruses, opsonize them for phagocytosis, or activate the complement
system.

- Cytotoxic T Cell Activation: Cytotoxic T cells recognize viral antigens presented by MHC class I
molecules on infected cells and induce apoptosis (cell death) of these cells through the release
of perforin and granzymes.

3. Viral Evasion of Immune Responses

Viruses have evolved various mechanisms to evade the immune system and these include

- Antigenic Variation: this refers to frequent mutations in viral proteins (e.g., influenza virus
hemagglutinin) prevent recognition by antibodies.

- Inhibition of Antigen Presentation: Some viruses (e.g., herpesviruses) downregulate MHC

molecules, preventing the activation of cytotoxic T cells.

- Immune Modulation: this Involves the production of viral proteins that inhibit interferon
signaling or other immune pathways.

- Latency: Some viruses (e.g., HIV and herpesviruses) can enter a latent state, evading immune
detection by reducing viral gene expression.

LABORATORY TECHNIQUES IN VIROLOGY

Laboratory techniques in virology are essential for the isolation, identification, and
characterization of viruses, as well as for studying their biology and interactions with host cells.
Here are some of the key techniques used in virology labs:

1. Virus Isolation and Cultivation

i. Cell Culture:

a. Primary Cell Culture: Cells are directly taken from living tissues and cultured. They have a
limited lifespan.

b. Continuous Cell Lines: these are immortalized cells that can be cultured indefinitely (e.g.,
HeLa, Vero cells). HeLa cells are a continuous cell line derived from cervical cancer cells of a
patient, Henrietta Lacks, who died of cervical cancer, while Vero cells are a lineage of cell
cultures derived from the kidney epithelial cells of an African green monkey

The maintenance requires specific culture media (e.g., DMEM, RPMI) supplemented with serum
(e.g., fetal bovine serum) and antibiotics to prevent contamination.

ii. Embryonated Eggs: this is used for cultivating certain viruses (e.g., influenza, poxviruses).

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Inoculation of the virus is done into specific sites such as the allantoic cavity or chorioallantoic
membrane.

iii. Animal Models: Used for studying virus pathogenesis and immune responses, and to
develop and to develop and test for vaccines and antiviral therapies. These models provide vital
important insights into how viruss interact with their hosts and help predict human responses to
infections and treatments. E.g mice (for studying viruses such as influenza, Zika and herpes
viruses), guinea pigs (for studying viral hemorrhagic fevers and respiratory infections), pigs (for
studying influenza and other zoonotic viruses), rabbits (for studying papilloma virus) etc.

2. Detection and Quantification of Viruses

i. Plaque Assay: this measures the number of plaque-forming units (PFUs) in a virus sample.
The cells are infected with serial dilutions of the virus, and plaques are counted to determine
virus concentration.

ii. TCID50 (Tissue Culture Infective Dose 50%) assay: this estimates the virus titer required to
infect 50% of cell cultures. This is useful for viruses that do not form clear plaques.

iii. Hemagglutination Assay: this is based on the ability of some viruses (e.g., influenza) to
agglutinate red blood cells. Hemagglutination titer reflects the concentration of the virus.

3. Viral Protein Detection

i. Enzyme-Linked Immunosorbent Assay (ELISA):- Detects viral antigens or antibodies against

viruses in a sample. It utilizes enzyme-linked antibodies that produce a colorimetric signal.

ii. Western Blot:- Detects specific viral proteins. Proteins are separated by gel electrophoresis,
transferred to a membrane, and probed with specific antibodies.

iii. Immunofluorescence: Uses fluorescently labeled antibodies to detect viral proteins in

infected cells or tissues. Observed under a fluorescence microscope.

4. Viral Nucleic Acid Detection

i. Polymerase Chain Reaction (PCR): Amplifies specific DNA sequences for detection. Reverse
transcription PCR (RT-PCR) is used for RNA viruses.

ii. Next-Generation Sequencing (NGS): High-throughput sequencing for comprehensive analysis

of viral genomes. Used for virus discovery, mutation analysis, and epidemiology studies.

iii. In Situ Hybridization: Detects viral nucleic acids within tissues or cells. It uses labeled nucleic
acid probes complementary to the viral genome.

5. Serological Techniques

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i. Neutralization Test:- Measures the ability of antibodies to neutralize viral infectivity. Involves
incubating the virus with serum samples before infecting cell cultures.

ii. Hemagglutination Inhibition Assay: Detects antibodies that inhibit viral hemagglutination.
Useful for influenza virus serotyping.

INTRODUCTION TO TISSUE CULTURE

Tissue culture refers to the technique of maintaining and growing tissues, cells, or organs
outside of the organism in a controlled artificial environment. This method is widely used in
research, biotechnology, and medicine for various applications, including studying cellular
mechanisms, drug testing, and producing biological compounds.

Historical Background

- Wilhelm Roux (1885): Performed one of the first experiments involving the maintenance of
tissues outside the body.

- Ross Granville Harrison (1907): Successfully cultivated frog nerve cells, marking the beginning
of modern tissue culture techniques.

- Carrel and Burrows (1910s): Developed methods for long-term culture of animal cells.

- Alexis Carrel (1912): Demonstrated that cells could be kept alive indefinitely under the right
conditions.

Types of Tissue Culture

1. Organ Culture: helps to maintain the architecture and functionality of the tissue and it is used
for studying organ development and function.

2. Explant Culture: these are small pieces of tissue are cultured to observe cell migration and
growth.

3. Cell Culture:

- Primary Cell Culture: Derived directly from tissues, having a finite lifespan.

- Secondary (or Continuous) Cell Lines: Derived from primary cultures or tumors, capable of
indefinite growth (e.g., HeLa, CHO cells).

Requirements for Tissue Culture

i. Culture Media: Provides essential nutrients, growth factors, and hormones. It is a commonly
used media include DMEM (Dulbecco’s Modified Eagle Medium), RPMI-1640, and MEM
(Minimum Essential Medium).

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- Supplements: Fetal bovine serum (FBS), antibiotics, and specific growth factors.

ii. Physical Environment: it is usually maintained at 37°C for mammalian cells. It is buffered to
maintain pH around 7.2-7.4 and 5-10% CO2 atmosphere to maintain the pH of the medium.

Applications of Tissue Culture in Virology

1. Virus Isolation and Propagation

Δ Culturing Viruses: Tissue culture techniques are used to isolate and propagate viruses from
clinical samples. Virus isolation involves inoculating cultured cells with a sample suspected of
containing a virus, followed by monitoring for cytopathic effects (CPE) or other indicators of
viral infection. Commonly used cell lines for virus isolation include Vero cells (for many viruses),
MDCK cells (for influenza virus), and HeLa cells (for various human viruses).

Δ Virus Stock Preparation: High-titer virus stocks are prepared by infecting cell cultures,
harvesting the virus-containing supernatant, and often purifying the virus. These stocks are
crucial for research, vaccine production, and diagnostic reagent development.

2. Vaccine Development

Δ Production of Viral Vaccines: Cell culture systems are used for the production of viral
vaccines. For example, influenza vaccines are produced using MDCK or Vero cells.

Δ Attenuation and Inactivation: Live-attenuated vaccines are developed by passaging the virus
in cell culture under conditions that attenuate its virulence. Inactivated vaccines are produced
by growing the virus in culture and then inactivating it with chemicals or heat.

Δ Vaccine Testing: Tissue cultures are used for preclinical testing of vaccine efficacy and safety.
Immunogenicity studies in vitro help predict the vaccine's ability to elicit an immune response.

3. Antiviral Drug Screening

High-Throughput Screening: Tissue culture systems enable high-throughput screening of

potential antiviral compounds. Infected cell cultures are treated with variocations of tissue in
virology

4. Pathogenesis and Host-Virus Interactions

Δ Studying Viral Life Cycles: Tissue culture allows detailed examination of the viral life cycle,
including attachment, entry, replication, assembly, and egress. Insights gained from these
studies can lead to the identification of new therapeutic targets.

Δ Host Cell Responses: Researchers study the host cell's response to viral infection, including
changes in gene expression, signaling pathways, and immune responses. The understanding of

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these interactions can reveal mechanisms of viral pathogenesis and host defense.

5. Diagnostic Assay Development: these assays assist in Viral Detection and Quantification
which include;

Δ Tissue culture techniques are used to develop and validate diagnostic assays for detecting
viral infections. These assays include immunofluorescence, ELISA, and PCR-based methods.

Δ Serological Testing: Cell cultures are used to produce viral antigens for serological tests that
detect antibodies against viruses in patient samples.

Δ Neutralization Assays: Tissue culture-based neutralization assays measure the ability of

antibodies to neutralize viral infectivity, important for evaluating immune responses and vaccine
efficacy.

6. Gene Therapy and Viral Vectors

Δ Production of Viral Vectors: Tissue culture systems are used to produce viral vectors for gene
therapy. Examples include adenoviruses, lentiviruses, and adeno-associated viruses (AAVs).

Δ Gene Delivery Studies: Tissue culture models are used to study the efficiency and specificity
of gene delivery by viral vectors. These studies help optimize viral vectors for therapeutic use.

7. For Basic Virology Research

Δ Genetic Studies: Genetic manipulation of viruses in tissue culture helps researchers

understand viral gene functions and regulatory mechanisms. For example, CRISPR/Cas9 and
RNA interference are used to knock out or knock down specific viral genes.

Δ Evolution and Adaptation: Tissue culture allows the study of viral evolution and adaptation to
different cell types and environmental conditions. Serial passaging of viruses in culture can lead
to the emergence of mutants with altered properties.

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 TUTORIAL QUESTIONS

1. What are viruses?

2. Explain 3 ways in which viruses can be transmitted

3. How would you describe the structure of viruses (b) Explain the viral replication cycle

4. Define the following terms:

(a) Incubation period (b) prodromal period (c) acute phase (d) convalescence period (e) chronic
phase (f) latent infection (g) incidence (h) endemic (i) pandemic (j) epidemic

5. Write short notes on (i)Herpes Simplex Virus (ii) Varicella-Zoster Virus (iii) Epstein Barr Virus

6. Write a short note on virus isolation and culture

7. Outline 5 applications of tissue culture in virology

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