This document discusses various methods for laboratory diagnosis of viral infections. It begins with an overview of the viral pathogenesis process, from attachment and entry into host cells, to replication of viral components, assembly, and release of new virus particles. The document then covers direct detection methods like electron microscopy, immunofluorescence microscopy, and light microscopy to identify inclusion bodies. It discusses serological tests to detect viral antigens or antibodies. Molecular methods like nucleic acid probes and PCR are mentioned. Isolation methods using animal inoculation, embryonated egg cultures, and tissue cultures are described in detail. The document provides a comprehensive overview of approaches for laboratory diagnosis of viral diseases.
Serological test for virus identificationPlock Ghosh
This presentation consist of detailed study of serological method of virus identification. Basically ELISA is vastly used for virus detection. Western blot method is used for HIV identification.
Serological test for virus identificationPlock Ghosh
This presentation consist of detailed study of serological method of virus identification. Basically ELISA is vastly used for virus detection. Western blot method is used for HIV identification.
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Adenoviridae is a group of medium sized, non-enveloped, double stranded DNA viruses that replicate and produce disease in the eye and in the respiratory, gastrointestinal and urinary tracts;
Poxviruses are brick or oval-shaped viruses with large double-stranded DNA genomes. Poxviruses exist throughout the world and cause disease in humans and many other types of animals. Poxvirus infections typically result in the formation of lesions, skin nodules, or disseminated rash.
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Adenoviridae is a group of medium sized, non-enveloped, double stranded DNA viruses that replicate and produce disease in the eye and in the respiratory, gastrointestinal and urinary tracts;
Poxviruses are brick or oval-shaped viruses with large double-stranded DNA genomes. Poxviruses exist throughout the world and cause disease in humans and many other types of animals. Poxvirus infections typically result in the formation of lesions, skin nodules, or disseminated rash.
lab diagnosis of viral infections - mayuri.pptxDrmayuribhise
T.M. River, 1937
Modified from Koch’s Postulates (proof of bacterial diseases)
Isolate virus from diseased hosts.
Cultivation of virus in host cells.
Proof of filterability.
Production of a comparable disease when the cultivated virus is used to infect experimental animals.
Reisolation of the same virus from the infected experimental animal.
Detection of a specific immune response to the virus.
Much more expensive and difficult to study animal viruses than bacteriophages
Cultivation in host cells
Living animal
Embryonated chicken eggs
Cell or tissue culture (= in vitro)
Over 60% of all infectious disease cases seen by a physician are due to viral infections.
Quality of patient specimens and their transport to the laboratory is importantViral Diagnostics in the Clinical Laboratory
Types of specimens:-
Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum
Eye infections: throat and Conjunctival swab/scraping
Gastrointestinal tract infections: stool and rectal swabs
Vesicular rash: vesicle fluid, skin scrapings
Maculopapular rash: throat, stool, and rectal swabs
CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid
Genital infections: vesicle fluid or swab
Urinary tract infections: urine
Blood borne infections: blood
Sterile, leak proof container
Minimal interval
Transport media
Viral infusion broth (VIB)
Sucrose-phosphate-glutamate (SPG)
Storage temperature:
4 deg C for up to 96 hours
Minus 70 deg C beyond 96 hours
Repeated cycles of freezing and thawing to be avoided
106 virus particles per ml required for visualization,
50,000 - 60,000 magnification normally used.Specimens are negatively stained by Potassium phosphotungstate and scanned under EM
Viruses may be detected in the following specimens.
Virus particles are detected and identified on the basis of morphology.
A) Shape
Rabiesvirus –bullet shaped
Rotavirus –Cart wheel
Coronavirus –petal shaped peplomers
Adenovirus –space vehicle shaped
Astrovirus ---Star shaped
B) Direct detection from specimens
For viruses that are difficult to cultivate ,EM can be used as primary tool for diagnosis
Faeces Rotavirus, Adenovirus
Norwalk like viruses
Astrovirus, Calicivirus
Vesicle Fluid HSV
VZV
Skin scrapings papillomavirus, orf
molluscum contagiosum
As an alternative to tissue culture
As tissues culture is time consuming and technically demanding ,EM is used as an alternative :-
1) Vesicular rashes –HSV and VZV detection from vesicular fluid
2) Meningitis—Detection of enterovirus and mumps from CSF.
Virus detection from tissue cultures EM can be used for detection of viral growth in tissue culture
The sensitivity and specificity of EM can be improved by adding specific antiviral antibody to the specimen to aggregate the virus particles which can be centrifuged
The sediment is negatively stained and viewed under EM
Direct immumofluroscence
Virology is the scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host cells for reproduction, their interaction with host organism physiology and immunity, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy
Virus is an obligate intracellular parasite which infects human beings, lower animals, insects, plants, bacteria and fungus. Viruses of medical importance to humans comprise of seven families of DNA viruses and 14 families of RNA viruses. Laboratory diagnosis of viral infections is continuously being refined to accelerate the process of identification of viruses. Because of the expense & the delay involved in obtaining a definitive virological diagnosis, discrimination in their use has to be done
serological test of HIV/ AIDS and their application.pptxDesalegn Ashenafi
I am graduated BSc in medical laboratory science with very great distinction and awarded gold medal for academic achievement.currently I am graduate assistant at Salale university and pursuing my master degree in medical microbiology at Jimma university . I want to conduct researches and give lectures on different areas of infectious disease, medical microbiology, diagnostic techniques
The earliest indications of the biological nature of viruses came from studies in 1892 by the Russian scientist Dmitry I. Ivanovsky and in 1898 by the Dutch scientist Martinus W. Beijerinck.
Beijerinck first surmised that the virus under study was a new kind of infectious agent, which he designated contagium vivum
fluidum, meaning that it was a live, reproducing organism that differed from other organisms.
Both of these investigators found that a disease of tobacco plants could be transmitted by an agent, later called tobacco mosaic virus, passing through a minute filter that would not allow the passage of bacteria.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
4. Adsorption/Attachment
• The viruses have attachment sites on their
envelopes or capsid proteins that bind to the
complementary receptor sites present on the
host cell surface.
HIV: surface glycoprotein gp 120 binds to CD4
molecules on the host cells.
lnfluenza: Viral hemagglutinin (an envelope
protein) binds specifically to gp receptors present
on the surface of respiratory epithelium.
5. Penetration
1. Phagocytosis (or viropexis):
It occurs through receptor mediated endocytosis resulting in the
uptake of virus particles within the endosomes of the host
cytoplasm.
2. Membrane fusion:
Some enveloped viruses (HIV) enter by fusion of their envelope
proteins with the plasma membrane of the host cell so that only
the nucleocapsid enters into the cytoplasm, whereas the viral
envelope remains attached to the host cell membrane.
3. Injection of nucleic acid:
Bacteriophages (viruses that infect bacteria) cannot penetrate
the rigid bacterial cell wall, hence only the nucleic acid is
injected; while the capsid remains attached to the cell wall.
6. • Uncoating
By the action of lysosomal enzymes of the host cells,
viral capsid gets separated and the nucleic acid is
released into the cytoplasm.
Absent in bacteriophages
• Biosynthesis
Components synthesized:
Nucleic acid
Capsid protein
Enzymes- required for various stages of viral replication.
Regulatory proteins- to shut down host cell metabolism.
In DNA viruses- DNA replication occurs in the nucleus
except in poxviruses
In RNA viruses- RNA replication occurs in cytoplasm
except in retroviruses and orthomyxoviruses
7. • Assembly
Viral nucleic acid and proteins are packaged
together to form progeny viruses (nucleocapsids)
Assembly take place in the host cell nucleus or
cytoplasm.
• Maturation
Maturation of daughter virions take place either in
the host cell nucleus or cytoplasm or membranes
(Golgi or Endoplasmic reticulum or Plasma
membrane)
8. Release
• Release of daughter virions occur either by:
1. Lysis of the host cells: Non enveloped viruses and
bacreriophages
2. Budding through host cell membrane:
• by enveloped viruses, during budding they
acquire a part of the host cell membrane to form
the lipid part of their envelopes.
• Envelope is acquired either from plasma
membrane (influenza virus) or from nuclear
membrane (e.g. herpesviruses).
• Viral glycoproreins are then inserted into the
envelopes.
9.
10. Transmission and spread of viruses
Mode of transmission Produce local infection at
the portal of entry
Spread to distant sites
from the portal of entry
Respiratory route (most
common route)
Produce respiratory
infection
Influenza virus
Parainfluenza virus
Respiratory syncytial virus
(RSV)
Rhinovirus
Adenovirus
Coronavirus
Herpes simplex virus (HSV)
Measles v irus
Mumps virus
Rubella virus
Varicella·zoster virus
Cytomegalovirus (CMV)
Parvovirus
Smallpox virus
Oral route Produce gastroenteritis
Rotavirus
Adenovirus 40,41
Calicivirus
Astrovirus
Poliovirus
Coxsackie virus
Hepatitis virus - A and E
Cytomegalovirus
Epstein-Barr virus (EBV)
Cutaneous route Produce skin lesions
Herpes simplex virus (HSV)
Human papillomavirus (HPV)
Molluscum contagiosum virus
Herpes simplex virus
11. • Depend up on the site of infection -
Throat swab, nasopharyngeal swab, bronchial lavage
Blood
Bone marrow
Rectal swab, stool
Urine
Sterile body fluid
Tissues
CSF
Serum
• Collection swab is made of Dacron
• Specimen transportion in VTM
Specimen
12. • Viral diagnosis
Direct Detection Detection of Molecular Isolation
Demonstration of viral Specific Methods of Virus
of Virus antigens Antibodies
Electron microscopy ELISA ELISA Nucleic acid probe Animal inoculation
lmmunoelectron microscopy direct IF, ICT PCR Embryonated egg inoculation
Fluorescent microscopy ICT Flow through assays Tissue cultures:
Light microscopy Flow through assays HA/HAI, Organ culture
CFT Explant culture
Histopathological staining Neutralization test Cell line culture
To demonstrate inclusion bodies Primary
lmmunoperoxidase staining Secondary and
Continuous cell lines
13. DIRECT DEMONSTRATION OF VIRUS
• Electron Microscopy - Specimens are negatively
stained by potassium phosphotungstate and scanned
under EM.
Rabies virus- Bullet shaped
Rotavirus- Wheel shaped
Coronavirus- Petal shaped peplomers
Adenovirus- Space vehicle shaped
Astrovirus- Star shaped peplomers
• Drawbacks: EM is highly expensive, has low
sensitivity with a detection threshold of 107
virions/mL. Specificity is also low
14. DIRECT DEMONSTRATION OF VIRUS
• lmmuno-electron Microscopy-
sensitivity and specificity of EM can be improved by
adding specific antiviral antibody to the specimen to
aggregate the virus particles which can be
centrifuged.
The sediment is negatively stained and viewed under
EM.
• Fluorescent Microscopy
Procedure: Specimen is mounted on slide, stained
with specific antiviral antibody tagged with
fluorescent dye and viewed under fluorescent
microscope
Diagnosis of rabies virus antigen in skin biopsies,
adenovirus from corneal smear of infected patients.
Syndromic approach: Rapid diagnosis of respiratory
infections (caused by influenza virus, rhinoviruses, respiratory syncytial
virus, adenoviruses and herpesviruses).
15. Light Microscopy
• Inclusion bodies:
Histopathogical staining of tissue sections used for
detection of inclusion bodies.
e.g. Negri bodies detection in brain biopsies of
patients or animals died of rabies
• lmmunoperoxidase staining:
Tissue sections/ cells coated with viral antigens
are stained using antibodies tagged with horse
radish peroxidase following which
Hydrogen peroxide and a coloring agent
(benzidine derivative) are added
Color complex formed can be viewed under under
microscope.
16. INCLUSION BODY
• They are the aggregates of virions or viral proteins and
other products of viral replication that confer altered
staining property to the host cell.
• They have distinct size, shape, location and staining
properties by which they can be demonstrated in virus
infected cells under the LM.
• Characteristic of specific viral infections.
• lntracytoplasmic IB:
Generally acidophilic
Seen as pink structures when stained with Giemsa or eosin
methylene blue stains
e.g. most poxviruses and rabies
• lntranuclear IB:
Basophilic in nature.
Cowdry (1934) had classified them into
• Cowdry type A inclusions- variable in size and have granular
appearance.
• Cowdry type B inclusions- more circumscribed and multiple
18. DETECTION OF VIRAL ANTIGENS
• Test used for detection of viral antigens in
serum and other samples are ELISA, ICT, flow
through assays etc.
HBsAg and HBeAg antigen detection- for hepatitis
B virus infection from serum
NS1 antigen detection- for dengue virus infection
from serum.
p24 antigen detection- for HIV infected patients
from serum.
Rotavirus antigen detection- from diarrheic stool.
CMV specific pp65 antigen detection- from serum
19. DETECTION OF VIRAL ANTIBODIES
• Most commonly used method in diagnostic virology
• Conventional Diagnostic Techniques- less commonly
used now a day. Examples
Heterophile agglutination test (e.g Paul-Bunnell rest for
Epstein-Barr virus).
Hemagglutination inhibition (HAI) test for influenza virus
and arbovirus infection.
Neutralization test- for poliovirus and arbovirus infections
CFT- for poliovirus, arbovirus and rabies virus infections
• Newer Diagnostic Formats -ELISA, ICT, flow through
assays example:
Anti-HBc, Anti-HBs and Anti-HBe antibodies for Hepatitis B
infection.
Anti-Hepatitis C antibodies
Antibodies against HIV-1 and HIV-2 antigens
Anti-Dengue IgM/IgG antibodies
20. MOLECULAR METHODS
• More sensitive, specific and yield quicker results
than culture.
• Nucleic Acid Probe-
An enzyme or radio-labelled nucleic acid sequence
complementry to a part of nucleic acid sequence of
the target virus.
Added to the clinical specimen- hybridizes to the
corresponding part of viral nucleic acid.
Both DNA and RNA probes are commercially available
• Polymerase Chain Reaction
• Reverse Transcriptase PCR (RT-PCR)
• Real Time PCR
21. ISOLATION OF VIRUS
• Viruses cannot be grown on artificial culture
media.
• They are cultivated by
Animal inoculation-
• Infant (suckling) mice are used.
• Specimens are inoculated by intracerebral or
intraperitoneal routes.
• Eg- intracerebral inoculation of Coxsackie virus into
suckling mice-
• Coxsackie-A virus produces flaccid paralysis
• Coxsackie-B virus produces spastic paralysis
Embryonated egg inoculation or
Tissue cultures.
22. Embryonated Egg Inoculation
• Embryonated hen’s eggs are used
• Specimens inoculated into embryonated 7 to
12 days old hen's eggs
• Incubated for 2-9 days.
• Routes of inoculation
Yolk Sac Inoculation
Amniotic Sac
Allantoic Sac
Chorioallantoic Membrane
24. Embryonated Egg Inoculation
• Yolk Sac Inoculation
Arboviruses- e.g. Japanese B encephalitis virus
Saint Louis encephalitis virus
West Nile virus and
Some bacteria such as Rickettsia, Chlamydia and
Haemophilus ducreyi
• Amniotic Sac
Primary isolation of the influenza virus
Viral growth measured by detection of
hemagglutinin antigens in amniotic fluid
25. Embryonated Egg Inoculation
• Allantoic Sac
Used for yield of viral vaccines like- influenza
vaccine, yellow fever (17D) vaccine and Rabies
(Flury strain) vaccine.
• Chorioallantoic Membrane (CAM)
Poxviruses, HSV and other viruses
Produce visible lesions called as pocks on CAM
Each pock derived from a single virion
26. Tissue Cultures
• Tissue culture - 3 types
• Organ culture:
For certain fastidious viruses that have affinity to
specific organs.
e.g. tracheal ring culture for isolation of corona virus
• Explant culture:
Obsolete now
Fragments of minced tissue can be grown as ‘explants’
e.g. Adenoid explants used for adenoviruses.
• Cell line culture:
Currently used method
Types - Primary cell lines, Secondary or diploid cell
lines, Continuous cell lines
27. Preparation of the Cell Lines
• Tissues are completely dissociated into individual cells
and dispensed in tissue culture flasks containing viral
growth medium
• Digested by- treatment with proteolytic enzymes
(trypsin or collagenase) followed by mechanical
shaking
• Viral growth medium: Contains balanced salt solution,
essential amino acids, vitamins, salts and glucose
supplemented by 5- 10% of fetal calf serum, antibiotics
and phenol red. pH of 7.2 to 7.4
• lncubation:- Tissue culture flasks are incubated
horizontally in presence of CO2 either as a stationery
culture or as a roller drum culture.
• Monolayer sheet formation: On incubation, the cells
adhere to glass surfaces of the flask and divide to form
a confluent monolayer sheet of cells within a week.
28. Cell lines
1. Primary cell lines
Derived from normal cells freshly taken from the organs and cultured.
Capable of very limited growth in culture, maximum up to 5-10
divisions.
Maintain a diploid karyosome.
examples –
• Monkey kidney cell line- useful for isolation of myxoviruses,
enteroviruses and adenoviruses
• Human amnion cell line
• Chick embryo cell line
2. Secondary or diploid cell lines:
Derived from the normal host cells and they maintain the diploid
karyosome
Divide maximum up to 10-50 divisions
Common examples:
• Human fibroblast cell line: CMV
• MRC-5 and WI-38 (human embryonic lung cell strain):- HSV, VZV,
CMV, adenoviruses, and picornaviruses also for vaccine for rabies,
chickenpox, hepatitis-A and MMR vaccines
29. Cell lines
3. Continuous Cell Lines
Derived from cancerous cell lines, hence are immortal &
capable of indefinite growth.
Possess altered haploid chromosome.
Easy to maintain in the laboratories by serial sub culturing
for indefinite divisions.
Examples
• HeLa cell line (Human carcinoma of cervix cell line).
• Hep-2 cell line (Human epithelioma of larynx cell line)-
widely used for RSV, adenovairuses and HSV.
• KB cell line (Human carcinoma of nasopharynx cell line).
• McCoy cell line (Human synovial carcinoma cell line)- useful
for isolation of viruses as well as Chlamydia .
• Vero cell line (Vervet monkey kidney cell line)-used for rabies
vaccine production.
• BHK cell line (Baby hamster kidney cell line)
30. DETECTION OF VIRAL GROWTH IN THE CELL CULTURES
• Methods used are
Cytopathic Effect (CPE)
Viral Interference
Hemadsorption
Direct immunofluorescence Assay
lmmunoperoxidase Staining
Electron Microscopy
Viral Genes Detection
31. Cytopathic Effect
• Morphological change produced by the virus
in the cell Iine, detected by light microscope.
• The type of CPE is unique for each virus and
that helps for their presumptive identification
32. Viral Interference
• The growth of a non-CPE virus in cell culture
can be detected by the subsequent challenge
of the cell line with a known CPE virus.
• Viral interference- The growth of the first virus
would inhibit infection by the second virus.
• For example, rubella is a non-CPE virus but
prevents the replication of enteroviruses.
33. • Hemadsorption-
The process of adsorption of erythrocytes to the
surfaces of infected cell lines cells is known as
hemadsorption.
Hemagglutinating viruses (e.g; influenza virus)
when grown in cell lines, they produce
hemagglutinin antigens which are coated on the
surface of the cell lines and detected by adding
guinea pig erythrocytes to the cultures.
• Direct immunofluorescence Assay
Virus infected cells are mounted on a slide and
stained with specific antibodies tagged with
fluorescent dye
Viewed under fluorescent microscope for the
presence of viral antigens on the surface of
infected cells.
34. • lmmunoperoxidase Staining
Cells coated with viral antigens are stained by
immunoperoxidase tagged specific antibodies and
viewed under Light microscope.
• Electron Microscopy
Viruses can also be demonstrated in infected cell
lines by EM
• Viral Genes Detection
By using PCR or nucleic acid probes