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Category of Sample
• Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab),
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Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material
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Microscopy Identification
• Light microscopy
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Light microscopy
6 • Cell death
Cell rounding Degeneration Aggregation
Loss of attachments to substrate
• Characteristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chromatin
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Fluorescent microscopy
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Electron microscopy
• Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus.
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Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material
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Viral isolation and Identification
• Viral Growth and Cell culture • Viral Detection
• Viral Identification
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Systems for the Propagation of
Viruses
• People
• Animals: cows, chickens, mice,rats, suckling mice • Embryonated eggs
• Organ and tissue culture Organ culture
Primary tissue culture Cell lines: diploid
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Viral detection
• CPE
• Hemadsorption • Interfere
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TCID50
(Tissue culture infective dose)
• TCID50 is defined as that dilution of virus
which will cause CPE in 50% of a given batch of cell culture
• TCID50= log10 of highest dilution giving 100%CPE +1/2 –
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Viral identification
• Complement fixation （
• Hemagglutination inhibition • Neutralization
• Immunofluorescence ( direct or indirect) • Latex agglutination
• In situ EIA • ELISA
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Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)
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Detection of viral proteins
( antigens and enzymes)
• Antigen detection ( ELISA, RIA, Western blot) • Hemagglutination and hemadsorption
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Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)]
• Detection of viral genetic material
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Detection of viral genetic material
• PCR ( Polymerase chain reaction)
• RT-PCR (Reverse transcriptase polymerase chain reaction)
• Southern （ DNA), Northern(RNA), and dot blots • DNA genome hybridization in situ(cytochemistry)
• Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis)
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Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material
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Serologic procedures
• If the antibody titer in the convalesent-phase serum sample is at least 4-fold higher than the
titer in the acute-phase serum sample, the patient is considered to be infected.
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Serologic procedures
• Complement fixation （
• Hemagglutination inhibition • Neutralization
• Immunofluorescence ( direct or indirect) • Latex agglutination
• In situ EIA • ELISA
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Viruses Diagnosed by Serology
• Epstein-Barr virus • Rubella virus
• Hepatitis A, B, C, D, and E viruses • HIV
• Human T-cell Leukemia virus
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Prevention
Prevention
•
Successes of the Past
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Active immunization
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Overview of Active immunization
• Active immunization - administration of
antigen resulting in production of a specific immune response with immunologic
memory. Response may be cellular or humoral or both.
Natural immunity - to diseases you have
caught and successfully fought
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Attributes of a good vaccine
• Ability to elicit the appropriate immune response for the particular pathogen
• Long term protection ideally life-long
• Safety vaccine itself should not cause disease • Stable retain immunogenicity, despite adverse
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LIVE VACCINES
•
Live attenuated organism
•
Heterologous vaccines
•
Live recombinant vaccines
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Live attenuated organism
• Organisms whose virulence has been
artificially reduced by in vitro Culture under adverse conditions, such as reduced
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Heterologous vaccines
• Closely related organism of lesser virulence,which shares many antigens with the virulent organism. The vaccine strain replication in
the host and induces an immune response that cross reacts with antigens of the virulent organism.
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Live recombinant
• Vector31
•Both cell mediated immunity and antibody response •Activates all phases of immune system. Can get humoral IgG and local IgA
•Raises immune response to all protective antigens. Inactivation may alter antigenicity.
•More durable immunity; more cross-reactive
•Immunity is long lived •Single dose
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Advantages of Attenuated
Vaccines 2-2
• Low cost
• Quick immunity in majority of vaccinees
• In case of polio and adeno vaccines, easy administration
• Easy transport in field
• Can lead to elimination of wild type virus from
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Disadvantages of Live Attenuated Vaccine
• Mutation; reversion to virulence (often frequent)
•Spread to contacts of vaccinee who have not
consented to be vaccinated (could also be an
advantage in communities where vaccination is not 100%)
• Spread vaccine not standardized--may be back-mutated
• Poor "take" in tropics
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Killed vaccines
• The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not
infectious and are therefore relatively safe. However, they are usually of lower
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Killed vaccines
• Inactivated organism: rabies virus; epidmic type B encephalitis virus.
• Subunit Vaccines: Influenza virus( HA and NA)
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Advantages of inactivated
vaccines
• Gives sufficient humoral immunity if boosters given
• No mutation or reversion
• Can be used with immuno-deficient patients • These vaccines tend to be able to
withstand more adverse storage
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Disadvantages of inactivated
vaccines
• Many vaccinees do not raise immunity • poor, only antibody, no cell immediated
immune response
• response is short-lived and multiple doses are needed
• No local immunity (important)
• Inactivated, therefore can not replicate in the host and cause disease
• Failure in inactivation and immunization with virulent virus
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New Methods
Selection of attenuated virus strain
• Varicella • Hepatitis A
Use monoclonal antibodies to select for virus with altered surface receptor
• Rabies • Reo
Use mutagen and grow virus at 32 degrees. Selects for
temperature-sensitive virus. Grows in upper respiratory tract but not lower
• ‘flu (new vaccine)
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New Methods
Passage progressively at cold temperatures TS mutant in internal proteins
40 PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS Attenuated Donor Master Strain New Virulent Antigenic Variant Strain
X
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New Methods
Deletion mutants
• Suppression unlikely (but caution in HIV)
• Viable but growth restrictions
Problems
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New Methods
• Recombinant DNA
•Single gene (subunit)
S-antigen mRNA
cDNA
Express plasmid
S-antigen mRNA protein
Hepatitis B vaccine
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Single gene (subunit) - problems
• Surface glycoprotein poorly soluble -
deletion?
• Poorly immunogenic
• Post-translational modifications
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Single gene (subunit) in
expression vector
Vaccinate with live virus
Canary Pox
• Infects human cells but does not replicate
• Better presentation
• CTL response
Vaccinia
Attenuated Polio
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New Methods
Chemically synthesized peptide
•
malaria
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antibody
New methods
Anti-idiotype vaccine
epitope
Antibody
with epitope
binding site
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antibody
Anti-idiotype vaccine
contMake antibody against antibody idiotype
Anti-idiotype antibody
Anti-idiotype
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Anti-anti-idiotype antibody
Anti-idiotype antibody
cont 2Use anti-idiotype antibody as injectable vaccine
Antibody to anti-idiotype antibody Binds and neutralizes virus Anti-idiotype antibody Anti-anti-idiotype antibody Anti-anti-idiotype antibody
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New Methods
New “Jennerian Vaccines”
• Live vaccines derived from animal strains of
similar viruses
• Naturally attenuated for humans
Rotavirus: Monkey Rota
80% effective in some human populations
Ineffective in others
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New Methods
New Jennerian Vaccines
Bovine parainfluenza Type 3 Bovine virus is:
• Infectious to humans
• Immunogenic (61% of children get good response)
• Poorly transmissable
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New Methods
Second Generation Jennerian Vaccines
Rotavirus
11 segments of double strand RNA Two encode:
• VP4 (hemagglutinin) • VP7 (glycoprotein)
Co-infect tissue culture cells reassortment •10 segments from monkey rotavirus
• 1 segment outer capsid protein of each of four major rotavirus strains Efficacy >80%
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Vaccines
•
1796 Jenner: wild type animal-adapted
virus
•
1800’s Pasteur: Attenuated virus
•
1996 DNA vaccines
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DNA vaccines
• DNA vaccines are at present experimental , but hold promise for future therapy since they evoke both humoral and cell-mediated
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DNA Vaccines
plasmid Muscle cell
Gene for antigen
Muscle cell expresses protein - antibody made
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DNA Vaccines
• Plasmids are easily manufactured in large amounts • DNA is very stable
• DNA resists temperature extremes so storage and transport are straight forward
• DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the
infectious agent
• By using the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are
processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than
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DNA Vaccines
• Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced
• The plasmid does not replicate and encodes only the proteins of interest
• No protein component so there will be no immune response against the vector itself
• Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the
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DNA Vaccines
Possible Problems
• Potential integration of plasmid into host genome leading to insertional mutagenesis
• Induction of autoimmune responses (e.g.
pathogenic anti-DNA antibodies)
• Induction of immunologic tolerance (e.g. where
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DNA Vaccines
DNA vaccines produce a situation that reproduces a virally-infected cell
Gives:
• Broad based immune response • Long lasting CTL response
Advantage of new DNA vaccine for flu:
CTL response can be against internal protein
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Adjuvants
• Certain substances, when administered
simultaneously with a specific antigen, will enhance the immune response to that
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Adjuvants in common use
• Aluminium salts
• Liposomes and immunostimulating complexes • Complet Freund’s adjuvant is an emulsion of
mycobacteria, oil and water • Incomplete Freund’s adjuvant • Muramyl di-peptide
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Possible action modes of adjuvant
• By trapping antigen in the tissues, thus
allowing maximal exposure to dendritic cells and specific T and B lymphocytes
• By activating antigen-presenting cells to secrete cytokines that enhance the
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Smallpox
•
Variolation
•1% v. 25%
mortality
•Life-long immunity
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Smallpox
Vaccination
• Jenner 1796 : Cowpox/Swinepox
• 1800’s Compulsory childhood vaccination
• 1930’s Last natural UK case
• 1940’s last natural US case
• 1958 WHO program
• October 1977: Last case
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Smallpox
• No animal reservoir• Lifelong immunity
• Subclinical cases rare
• Infectivity does
not precede overt symptoms
• One Variola serotype
• Effective vaccine
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polio
• Killed virus vaccine(Salk, 1954)
• Live attenuated oral polio vaccine( Sabin, 1957)
• The inactivated Salk vaccines is
recommended for children who are immunosuppressed.
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Small RNA virus Some drift…but not too far as non-viable
Sabin attenuated vaccine
~ 10 cases vaccine-associated disease per year • 50% vaccinees feces
• 50% contacts
• Vaccine-associated cases: revertants
• 1 in 4,000,000 vaccine infections paralytic polio • 1 in 100 of wt infections
Scandinavia: Salk dead vaccine
• No gut immunity
• Cannot wipe out wt virus
68 R ep o rt ed c as es p er 1 00 00 0 p o p u la ti o n 100 10 1 0.1 0.001 0.01
1950 1960 1970 1980 1990
Inactivated (Salk) vaccine
Oral vaccine
69 10000 1000 100 10 1 0 R ep o rt ed c as es
1950 1955 1960 1965 1970 1975
Killed (Salk) vaccine
Total cases
70 R ec ip ro ca l v ir us a nt ib o dy t ite r 512 128 32 8 2 1
Serum IgG Serum IgG
Serum IgM Serum IgM
Nasal and duodenal IgA Nasal IgA Serum IgA Serum IgA Duodenal IgA Days Vaccination Vaccination
48 96 48 96
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Sabin Polio Vaccine
Attenuation by passage in foreign hostMore suited to foreign environment and less suited to original host
Grows less well in original host
Polio:
• Monkey kidney cells
• Grows in epithelial cells
• Does not grow in nerves
• No paralysis
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Salk Polio Vaccine
•
Formaldehyde-fixed
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Polio Vaccine
Why use the Sabin vaccine?:
• Local immunity: Vaccine virus just like natural infection
• Stopping replication in G.I. Tract stops viral replication TOTALLY
• Dead Salk vaccine virus has no effect on gut replication
• No problem with selective inactivation
• Greater cross reaction as vaccine virus also has antigenic drift
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Measles
• Live attenuated virus grown in chick embryo fibroblasts, first introduced in the 1960’s.
• Etiology: Measles virus • Incubation: 8 to 12 days
• Clinical Manifestations: cough, coryza, conjunctivitis , erythematous maculopapular rash
fever ,Koplik Spots ,complictions include Encephalitis, Pneumonia, and SSPE
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Mumps
• Live attenuated virus developed in the 1960’s • MMR vaccine
• Etiology: Mumps Virus • Incubation: 16 to 18 days • Clinical Manifestations:
• swelling of the salivary glands
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rubella
• Live attenuated virus • Etiology: Rubella Virus • Incubation: 14 to 21 days
• Clinical Manifestations: Congenital , cataracts • patent ductus arteriosus , deafness mental
retardation , Postnatal mild disease , erythematous maculopapular rash , postauricular
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Hepatitis B
• Two vaccines are in current use: A serum derived vaccine
A recombinant vaccine • Etiology: Hepatitis B Virus
• Incubation: 120 days (average)
• Clinical Manifestations: jaundice ; anorexia • nausea and vomiting ; malaise
• complications include the development of a chronic carrier state with a high risk for Hepatocellular
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Hepatitis A
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Yellow fever
• The 17D strain is a live attenuated vaccine developed in 1937.
• It is a highly effective vaccine which is
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Rabies
No safe attenuated strain of rabies virus has yet been developed for human. Vaccines in current use include: a] The neurotissue vaccine
b] human diploid cell culture-derived vaccine, which is much safer.
There are two situation where vaccine is given: a] Post-exposure prophylaxis, followinf the bite of a rabid animal, Hyperimmune rabies globulin may also administered .
b] Pro-exposure prophylaxis is used for
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Influenza
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Varicella-Zoster virus
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Modes of immunization
• Passive immunization - administration of
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Natural
• Provides immunity for diphtheria, tetanus,
streptococcus, rubeola (red measles), rubella (German measles), mumps, polio, and
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Artificial
• Often used as antitoxins for things such as black widow spider and snake bites,
botulism, and tetanus. Important for some infectious diseases such as rabies, since it provides immediate protection rather than waiting the 7-10 days for a protective
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Immunoglobulin
• “Normal”Immune globulin
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“Normal”Immune globulin
Low titres of antibody to a wide range of human viruses
• Hepatitis A virus infection
• Parvovirus infection
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Hyper-immune globulin
---
high titres of antibody to particular viruses• Zoster immune globulin: prevention of varicella in immunocompromised children and neonates
• Human rabies immunoglobulin: post-exposure
prophylaxis in an individual who has been bitten by a rabid animal
• Hepatitis B immune globulin:non-immune individal who has been exposed to HBV
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Antiviral Therapy
• Antiviral chemotherapy • Interferon
• Gene therapy
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Antiviral chemotherapeutic Agents
• Antiviral drugs are available to treat only a few viral diseases.
• The reason for this is the fact that viral
replication is so intimately associated with the host cell that any drug that interferes
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Targets for chemotherapeutic
agents
• Attachment to host cell • Uncoating –(amantadine)
• Synthesis of viral mRNA-(interferon) • Translation of mRNA-(interferon)
• Replication of viral RNA or DNA- (nucleoside anologues)
• Maturation of new virus proteins-(protease inhibitors)
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Diseases for which effective
therapy is available
• AIDS:
Zidovudine （（（（ + Lamivudine （（（（ + protease inhibitors
• Influenza: Amantadine
• Herpes simplex virus: Acyclovir
• Varicella-Zoster virus: Acyclovir
• Cytomegalovirus : Gancyclovir （（（（ , Foscarnet （（ （
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Nucleotide analogues
• Nucleotide analogues competes with
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Interferon
• Direct antiviral effect ( prevents the infection of new cells) by a) degradation of viral
mRNA, and b) inhibition of protein synthesis • Enhancement of the specofic
immuneresponse by increasing the
expression of MHC class I molecules on the surface of infected cells, the interferons
increase the opportunity for specifif cytotoxic T cells to recognise and kill infected cells
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