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
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 virus-induced cell-cell fusion

Syncytia: multinucleated giant cells caused by

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
enzymes)] Detection of viral genetic material Serologic procedures

Viral isolation and Identification

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
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

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
enzymes)] Detection of viral genetic material Serologic procedures

Detection of viral genetic material

PCR ( Polymerase chain reaction) RT-PCR (Reverse transcriptase polymerase

chain reaction) SouthernDNA), 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
enzymes)] Detection of viral genetic material Serologic procedures

Serologic procedures
If the antibody titer in the convalesentphase 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

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

 Active immunization - administration of

Overview of Active immunization

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

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

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

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

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

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 raised in yeast
Express plasmid S-antigen mRNA cDNA

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

Make antibody against antibody idiotype

Anti-idiotype antibody mimics the epitope

antibody

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
1800s 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 Freunds adjuvant is an emulsion of mycobacteria, oil and water Incomplete Freunds 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 1800s Compulsory childhood vaccination 1930s Last natural UK case 1940s 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.

Polio Vaccine
Small RNA virus viable Some driftbut not too far as non-

Sabin attenuated vaccine

~ 10 cases vaccine-associated disease per year 50% vaccinees 50% contacts Vaccine-associated cases: revertants feces

1 in 4,000,000 vaccine infections

1 in 100 of wt infections Scandinavia: Salk dead vaccine

paralytic polio

No gut immunity

Reported cases per 100000 population

100

10

Inactivated (Salk) vaccine Oral vaccine

Cases per 100,000 population United States

0. 1
0.01 0.00 1 195
0

1960

1970

1980

1990

10000

Total cases Sweden and Finland

Killed (Salk) vaccine

Reported cases

1000

100

1 0 1 0 195 0

1955

1960

196 5

197 0

1975

512

Killed (Salk) Vaccine

Serum IgG

Live (Sabin) Vaccine

Serum IgG

Reciprocal virus antibody titer

128

32

Serum IgM

Serum IgM

Serum IgA

Nasal IgA Serum IgA

Nasal and duodenal IgA

1
Vaccination 4 8 96

Duodenal IgA
48 9 6

Days Vaccinatio

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

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 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

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

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

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)

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
NormalImmune globulin Hyper-immune globulin

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

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

Hyper-immune globulin --- high titres of antibody to particular

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

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:
Zidovudine+ Lamivudine+ protease inhibitors

Influenza: Amantadine Herpes simplex virus: Acyclovir Varicella-Zoster virus: Acyclovir Cytomegalovirus : Gancyclovir, Foscarnet Respiratory syncitial virus: Ribavirin

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