Laboratory Diagnosis in virus diseases

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Laboratory Diagnosis in virus diseases
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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), tissue ,brain
  biopsy, cerebrospinal fluid, et al.

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Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
  enzymes)
• Detection of viral genetic material
• Serologic procedures

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Microscopy Identification

• Light microscopy
• Fluorescent microscopy
• Electron microscopy

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Light microscopy

• Characteristic CPE
• Inclusion Bodies

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• Cell death
• Cell rounding
• Degeneration
• Aggregation
• Loss of attachments to substrate
• Characteristic histological changesinclusion
  bodies in the nucleus or cytoplasm, margination
  of chromatin
• Syncytia multinucleated giant cells caused by
  virus-induced cell-cell fusion

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Fluorescent microscopy

• Fluorescent-antibody staining

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Electron microscopy

• Direct detection Human rotavirus HAV HBV
  Smallpox virus Herpes virus.
• Immune Electron microscopy Human rotavirus HAV

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Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
  enzymes)
• Detection of viral genetic material
• Serologic procedures

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Viral isolation and Identification

• Viral Growth and Cell culture
• Viral Detection
• Viral Identification
• Interpretation of culture results

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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
• Tumor or (immortalized )cell line

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Viral detection

• CPE
• Hemadsorption
• Interfere
• Metabolize of cell

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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 100CPE
  1/2 (total number of test units showing CPE)/
  (number of test units per dilution)

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Viral identification

• Complement fixation
• Hemagglutination inhibition
• Neutralization
• Immunofluorescence ( direct or indirect)
• Latex agglutination
• In situ EIA
• ELISA
• RIA(radioimmuno

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Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
  enzymes)
• Detection of viral genetic material
• Serologic procedures

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Detection of viral proteins( antigens and
enzymes)

• Antigen detection ( ELISA, RIA, Western blot)
• Hemagglutination and hemadsorption
• Enzyme activities( reverse transcriptase)
• Protein patterns( 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
• Serologic procedures

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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)
• Restriction endonuclease cleavage patterns

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Laboratory Diagnosis

• Microscopy Identification
• Virus isolation and identification
• Detection of viral proteins( antigens and
  enzymes)
• Detection of viral genetic material
• Serologic procedures

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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.
• In certain viral diseases, the presence of IgM
  antibody is used to diagnose current infection
• Other nonspecific serologic tests are available

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Serologic procedures

• Complement fixation
• Hemagglutination inhibition
• Neutralization
• Immunofluorescence ( direct or indirect)
• Latex agglutination
• In situ EIA
• ELISA
• RIA

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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
• Arboviruses ( Encephalitis viruses)

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Prevention

• Successes of the Past
• Possibilities for the Future

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Active immunization

• Vaccines

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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
• Artificial immunity Vaccination(vaccines)

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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
  storage conditions prior to administration
• In-expensive

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LIVE VACCINES

• Live attenuated organism
• Heterologous vaccines
• Live recombinant vaccines
• Attributes live 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 temperature.

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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.
• Vaccinia virus /cowpox virus--- Variola virus

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Live recombinant

• Vector
• bovine vaccine
• BCG

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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

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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
  the community

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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
• Problem in immunodeficiency disease (may spread
  to these patients)

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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 immunogenicity and multiple
  doses may be needed to induce immunity. In
  addition, they are usually expensive to prepare.

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Killed vaccines

• Inactivated organism rabies virus epidmic type
  B encephalitis virus.
• Subunit Vaccines Influenza virus( HA and NA)
• Recombinant proteins HBV

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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 conditions,Sometimes better in
  tropics

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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
• Expense Expensive to prepare

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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)
• respiratory syncytial virus

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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
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New Methods

• Deletion mutants
• Suppression unlikely (but caution in HIV)
• Viable but growth restrictions
• Problems
• Oncogenicity in some cases (adeno, retro)

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New Methods
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Single gene (subunit) - problems

• Surface glycoprotein poorly soluble - deletion?
• Poorly immunogenic
• Post-translational modifications
• Poor CTL response

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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
• Being developed for anti-HIV vaccine

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New Methods

• Chemically synthesized peptide
• malaria
• poorly immunogenic

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New methods
Anti-idiotype vaccine
Virus
epitope
Antibody with epitope binding site
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Anti-idiotype vaccine cont
Make antibody against antibody idiotype
Anti-idiotype antibody mimics the epitope
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Anti-idiotype antibody cont 2
Use anti-idiotype antibody as injectable vaccine
Use as vaccine
Binds and neutralizes virus
Antibody to 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
• Due to differences in circulating viral serotypes

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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
• Phenotypicaly stable

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New Methods
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Vaccines

• 1796 Jenner wild type animal-adapted virus
• 1800s Pasteur Attenuated virus
• 1996 DNA vaccines
• The third vaccine revolution

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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 immunity, without
  the dangers associated with live virus vaccines

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DNA Vaccines
Gene for antigen
Muscle cell
plasmid
Muscle cell expresses protein - antibody made CTL
response
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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 purifying
  that protein and using it as an immunogen.

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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 pathogen. A CTL
  response also offers protection against diseases
  caused by certain obligate intracellular
  pathogens (e.g. Mycobacterium tuberculosis)

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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
  the expression of the antigen in the host may
  lead to specific non-responsiveness to that
  antigen)

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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
• In mice a nucleoprotein DNA vaccine is effective
  against a range of viruses with different
  hemagglutinins

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Adjuvants

• Certain substances, when administered
  simultaneously with a specific antigen, will
  enhance the immune response to that antigen.

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Adjuvants in common use

• Aluminium salts
• Liposomes and immunostimulating complexes
• Complet Freunds adjuvant is an emulsion of
  mycobacteria, oil and water
• Incomplete Freunds adjuvant
• Muramyl di-peptide
• Cytokines

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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 recruitment of
  antigen-specific T and B cells to the site of
  inoculation

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Smallpox
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Smallpox

• Variolation
• 1 v. 25 mortality
• Life-long immunity
• No drift or shift

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Smallpox
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Smallpox
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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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Polio Vaccine
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100
Inactivated (Salk) vaccine
Cases per 100,000 population United States
10
Oral vaccine
1
Reported cases per 100000 population
0.1
0.01
0.001
1950
1960
1990
1970
1980
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Total casesSweden and Finland
10000
Killed (Salk) vaccine
1000
Reported cases
100
10
1
0
1950
1955
1960
1965
1970
1975
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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

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Salk Polio Vaccine

• Formaldehyde-fixed
• No reversion

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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
• Life-long immunity

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Measles

• Live attenuated virus grown in chick embryo
  fibroblasts, first introduced in the 1960s.
• 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

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Mumps

• Live attenuated virus developed in the 1960s
• MMR vaccine
• Etiology Mumps Virus
• Incubation 16 to 18 days
• Clinical Manifestations
• swelling of the salivary glands
• complications include Meningitis, Orchitis,
  Encephalitis, and Deafness

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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
  lymphadenopathy transient polyarthralgias

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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 Carcinoma (liver cancer)

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Hepatitis A

• Formalin-inactivated , cell cultured-derived
  virus,

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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
  administered to residents in the tropics and
  travellers to endemic areas.

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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
  protection of those occupation puts them at risk
  of infection with rabies.

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Influenza

• New vaccines are produced every year

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Varicella-Zoster virus

• Not licensed vaccines

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Passive Immunisation
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Modes of immunization

• Passive immunization - administration of
  antibody-containing serum to provide immediate,
  but temporary protection. Doesn't activate a
  lasting specific immune response.

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Natural

• Provides immunity for diphtheria, tetanus,
  streptococcus, rubeola (red measles), rubella
  (German measles), mumps, polio, and others.

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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 response to develop from active
  immunization.

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Immunoglobulin

• NormalImmune globulin
• Hyper-immune globulin

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NormalImmune 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

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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 globulinnon-immune individal
  who has been exposed to HBV
• RSV immune globulin treatment of respiratory
  syncitial virus infections in the very young

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Antiviral Therapy
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Antiviral Therapy

• Antiviral chemotherapy
• Interferon
• Gene therapy
• Chinese Herbs

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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
  significantly with viral replication, is likely
  to be toxic to the host

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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)
• Budding , release

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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???
• Respiratory syncitial virus Ribavirin????

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Nucleotide analogues

• Nucleotide analogues competes with normal
  nucleotide for incorporation into viral DNA or
  RNA.

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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
• Chronic hepatitis B and C virus

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Chinese Herbs

• ??????????????
• ???