Transcript Detection of viral genetic material

Laboratory Diagnosis
in virus diseases
Category of Sample
• Blood, Urine, Stool, nasal washing,
nasal swab , throat swab, saliva ,
sputum, rectal swab, vesicle
fluid( scraping or swab), tissue ,brain
biopsy, cerebrospinal fluid, et al.
Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
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enzymes)]
Detection of viral genetic material
Serologic procedures
Microscopy Identification
• Light microscopy
• Fluorescent microscopy
• Electron microscopy
Light microscopy
• Characteristic CPE
• Inclusion Bodies
• Cell death
Cell rounding
Degeneration
Aggregation
Loss of attachments to substrate
• Characteristic histological changes:inclusion
bodies in the nucleus or cytoplasm, margination
of chromatin
• Syncytia: multinucleated giant cells caused by
virus-induced cell-cell fusion
Fluorescent microscopy
• Fluorescent-antibody staining
Electron microscopy
• Direct detection : Human rotavirus; HAV;
HBV; Smallpox virus; Herpes virus.
• Immune Electron microscopy: Human
rotavirus; HAV;
Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
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enzymes)]
Detection of viral genetic material
Serologic procedures
Viral isolation and Identification
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Viral Growth and Cell culture
Viral Detection
Viral Identification
Interpretation of culture results
Systems for the Propagation of
Viruses
• People
• Animals: cows, chickens, mice,rats, suckling
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mice
Embryonated eggs
Organ and tissue culture
Organ culture
Primary tissue culture
Cell lines: diploid
Tumor or （immortalized ）cell line
Viral detection
• CPE
• Hemadsorption
• Interfere
• Metabolize of cell
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 – (total number of test units
showing CPE)/ (number of test units per
dilution)
Viral identification
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Complement fixation：
Hemagglutination inhibition
Neutralization
Immunofluorescence ( direct or indirect)
Latex agglutination
In situ EIA
ELISA
RIA(radioimmuno
Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
•
•
enzymes)
Detection of viral genetic material
Serologic procedures
Detection of viral proteins
( antigens and enzymes)
• Antigen detection ( ELISA, RIA, Western
blot)
• Hemagglutination and hemadsorption
• Enzyme activities( reverse transcriptase)
• Protein patterns( electrophoresis )
Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
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enzymes)]
Detection of viral genetic material
Serologic procedures
Detection of viral genetic
material
• PCR ( Polymerase chain reaction)
• RT-PCR (Reverse transcriptase polymerase
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chain reaction)
Southern（DNA), Northern(RNA), and dot blots
DNA genome hybridization in situ(cytochemistry)
Electrophoretic mobilities of RNA for segmented
RNA viruses( Electrophoresis)
Restriction endonuclease cleavage patterns
Laboratory Diagnosis
• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
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enzymes)]
Detection of viral genetic material
Serologic procedures
Serologic procedures
• If the antibody titer in the convalesent-
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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.
In certain viral diseases, the presence of
IgM antibody is used to diagnose current
infection
Other nonspecific serologic tests are
available
Serologic procedures
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Complement fixation：
Hemagglutination inhibition
Neutralization
Immunofluorescence ( direct or indirect)
Latex agglutination
In situ EIA
ELISA
RIA
Viruses Diagnosed by
Serology
• Epstein-Barr virus
• Rubella virus
• Hepatitis A, B, C, D, and E viruses
• HIV
• Human T-cell Leukemia virus
• Arboviruses ( Encephalitis viruses)
Prevention
•
Successes of the Past
• Possibilities for the Future
Active immunization
Vaccines
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
 Artificial immunity – Vaccination(vaccines)
Attributes of a good vaccine
• Ability to elicit the appropriate immune
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response for the particular pathogen
Long term protection ideally life-long
Safety vaccine itself should not cause disease
Stable retain immunogenicity, despite adverse
storage conditions prior to administration
In-expensive
LIVE VACCINES
• Live attenuated organism
• Heterologous vaccines
• Live recombinant
vaccines
•
Attributes – live
Live attenuated
organism
• Organisms whose virulence has been
artificially reduced by in vitro Culture
under adverse conditions, such as reduced
temperature.
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.
• Vaccinia virus /cowpox virus--- Variola
virus
Live recombinant
• Vector
1. bovine vaccine
2. BCG
Advantages of Attenuated
Vaccines 2-1
•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
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 the community
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 backmutated
• Poor "take" in tropics
• Problem in immunodeficiency disease (may
spread to these patients)
Killed vaccines
• The organism is propagated in bulk, in
vitro, and inactivated with either betapropiolactone or formaldehyde. These
vaccines are not infectious and are
therefore relatively safe. However, they
are usually of lower immunogenicity and
multiple doses may be needed to induce
immunity. In addition, they are usually
expensive to prepare.
Killed vaccines
• Inactivated organism: rabies virus;
epidmic type B encephalitis virus.
• Subunit Vaccines: Influenza
virus( HA and NA)
• Recombinant proteins: HBV
Advantages of inactivated
vaccines
• Gives sufficient humoral immunity if
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boosters given
No mutation or reversion
Can be used with immuno-deficient
patients
These vaccines tend to be able to
withstand more adverse storage
conditions,Sometimes better in tropics
Disadvantages of inactivated
vaccines
• Many vaccinees do not raise immunity
• poor, only antibody, no cell
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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
Expense: Expensive to prepare
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)
• respiratory syncytial virus
New Methods
Passage progressively at cold temperatures
TS mutant in internal proteins
Can be re-assorted to so that coat is the strain
that is this years flu strain
PB2
PB1
PA
HA
NA
NP
M
NS
Attenuated Donor
Master Strain
Attenuated Vaccine
Strain: Coat of Virulent
strain with Virulence
Characteristics of
Attenuated Strain
X
PB2
PB1
PA
HA
NA
NP
M
NS
PB2
PB1
PA
HA
NA
NP
M
NS
New Virulent
Antigenic
Variant Strain
New Methods
Deletion mutants
• Suppression unlikely (but caution in HIV)
• Viable but growth restrictions
Problems
• Oncogenicity in some cases (adeno, retro)
New Methods
• Recombinant DNA
•Single gene (subunit)
Hepatitis B
vaccine
S-antigen
mRNA
cDNA
raised in yeast
Express
plasmid
S-antigen
mRNA
protein
Single gene (subunit) problems
• Surface
glycoprotein poorly soluble deletion?
• Poorly immunogenic
• Post-translational modifications
• Poor CTL response
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
Being developed for anti-HIV vaccine
New Methods
Chemically synthesized
peptide
• malaria
poorly immunogenic
New methods
Anti-idiotype vaccine
Virus
epitope
antibody
Antibody
with
epitope
binding site
Anti-idiotype vaccine cont
Antiidiotype
antibody
antibody
Make antibody
against antibody
idiotype
Anti-idiotype
antibody mimics
the epitope
Anti-idiotype antibody
cont 2
Use anti-idiotype antibody as
injectable vaccine
Anti-idiotype
antibody
Use as
vaccine
Binds and
neutralizes virus
Anti-anti-idiotype
antibody
Anti-anti-idiotype
antibody
Antibody to antiidiotype
antibody
Anti-anti-idiotype
antibody
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
Due to differences in circulating viral
New Methods
New Jennerian Vaccines
Bovine parainfluenza Type 3
Bovine virus is:
• Infectious to humans
• Immunogenic (61% of children get good
response)
• Poorly transmissable
•Phenotypicaly stable
New Methods
Second Generation Jennerian Vaccines
Rotavirus
11 segments of double strand RNA
Two encode:
• VP4 (hemagglutinin) Elicit neutralizing
• VP7 (glycoprotein) antibodies
Co-infect tissue culture cells
reassortment
•10 segments from monkey rotavirus
• 1 segment outer capsid protein of each of four major
rotavirus strains
Vaccines
• 1796 Jenner: wild type animaladapted virus
• 1800’s Pasteur: Attenuated virus
• 1996 DNA vaccines
The third vaccine revolution
DNA vaccines
• DNA vaccines are at present experimental ,
but hold promise for future therapy since
they evoke both humoral and cellmediated immunity, without the dangers
associated with live virus vaccines
DNA Vaccines
Gene
for
antigen
plasmid
Muscle cell
Muscle cell
expresses protein antibody made
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 purifying that
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 pathogen. A CTL response also offers
protection against diseases caused by certain obligate
intracellular pathogens (e.g. Mycobacterium
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 the expression of the antigen in the
host may lead to specific non-
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
In mice a nucleoprotein DNA vaccine is effective against a range
of viruses with different hemagglutinins
Adjuvants
• Certain substances, when administered
simultaneously with a specific antigen, will
enhance the immune response to that
antigen.
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
• Cytokines
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
recruitment of antigen-specific T and B
cells to the site of inoculation
Smallpox
Smallpox
• Variolation
•1% v. 25%
mortality
•Life-long
immunity
• No drift or shift
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
Smallpox
• No animal reservoir
• Lifelong immunity
• Subclinical cases rare
• Infectivity does
not precede overt symptoms
• One Variola serotype
• Effective vaccine
• Major commitment by governments
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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Polio Vaccine
Small RNA virus
viable
Some drift…but not too far as non-
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
• 1 in 100 of wt infections
Scandinavia: Salk dead vaccine
• No gut immunity
paralytic polio
Reported cases per 100000 population
100
10
Inactivated
(Salk)
vaccine
Oral
vaccine
Cases per
100,000
population
United States
1
0.
1
0.01
0.00
1 195
0
1960
1970
1980
1990
Total cases
Sweden and
Finland
Reported cases
10000
Killed
(Salk)
vaccine
1000
100
1
0
1
0
195
0
1955
1960
196
5
197
0
1975
Reciprocal virus antibody titer
512
Killed
(Salk)
Vaccine
Live
(Sabin)
Vaccine
Serum
IgG
Serum
IgG
128
32
Serum IgM
Serum
IgM
Serum
IgA
8
Nasal
IgA
Serum
IgA
2
Duodenal
IgA
Nasal and
duodenal IgA
1
Vaccination
4
8
96
Days Vaccinatio
48
9
6
Sabin Polio Vaccine
Attenuation by passage in foreign host
More 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
•Local gut immunity (IgA)
Pasteur rabies vaccine also attenuated
Salk Polio Vaccine
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Formaldehyde-fixed
• No reversion
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
• Life-long immunity
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
• Treatment: Supportive
Mumps
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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
complications include Meningitis, Orchitis,
Encephalitis, and Deafness
rubella
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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
lymphadenopathy transient polyarthralgias
Hepatitis B
• Two vaccines are in current use:
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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 Carcinoma (liver cancer)
Hepatitis A
• Formalin-inactivated , cell culturedderived virus,
Yellow fever
• The 17D strain is a live attenuated vaccine
developed in 1937.
• It is a highly effective vaccine which is
administered to residents in the tropics
and travellers to endemic areas.
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 .
Influenza
• New vaccines are produced every year
Varicella-Zoster virus
• Not licensed vaccines
Passive
Immunisation
Modes of immunization
• Passive immunization - administration of
antibody-containing serum to provide
immediate, but temporary protection. Doesn't
activate a lasting specific immune response.
Natural
• Provides immunity for diphtheria, tetanus,
streptococcus, rubeola (red measles),
rubella (German measles), mumps, polio,
and others.
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
response to develop from active
immunization.
Immunoglobulin
• “Normal”Immune globulin
• Hyper-immune globulin
“Normal”Immune globulin
Low titres of antibody to a wide
range of human viruses
• Hepatitis A virus infection
• Parvovirus infection
• Enterovirus infections (in
neonates)
• HIV-infected babies
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
• RSV immune globulin: treatment of respiratory
syncitial virus infections in the very young
Antiviral Therapy
Antiviral Therapy
• Antiviral chemotherapy
• Interferon
• Gene therapy
• Chinese Herbs
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
significantly with viral replication, is likely
to be toxic to the host
Targets for chemotherapeutic
agents
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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)
Budding , release
Diseases for which effective
therapy is available
• AIDS:
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Zidovudine叠氮胸苷+ Lamivudine拉米夫定+
protease inhibitors
Influenza: Amantadine
Herpes simplex virus: Acyclovir
Varicella-Zoster virus: Acyclovir
Cytomegalovirus : Gancyclovir更昔洛韦,
Foscarnet膦甲酸
Respiratory syncitial virus: Ribavirin利巴韦林
Nucleotide analogues
• Nucleotide analogues competes with
normal nucleotide for incorporation
into viral DNA or RNA.
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
Chinese Herbs
• 板蓝根、大青叶、苍术、艾叶。
• 双黄连