Diagnosis of viral infections/diseases

Dr F Noordeen Department of Microbiology Faculty of Medicine University of Peradeniya April 2013

Learning outcomes
1. State different methods used in diagnosis of viral infections 2. Briefly explain the limitations of each method 3. Outline the principles of diagnosis of viral infections in Sri Lanka Dengue Rapid assays for IgM / IgG and PCR for NA Hepatitis Rapid assays for antigen in hepatitis B and IgM in hepatitis A Influenza FAT for antigen, Rapid assays for antigen and PCR for NA Rabies Microscopy for inclusion bodies and FAT for antigen

Diagnosis of viral diseases

1. Identification of Virus
- See virus/virus mediated changes - Detect antigen or nucleic acid of virus - Grow virus

2. Identify host response to virus - Virus specific antibody detection

See virus
Intranuclear Inclusion bodies

Herpes simplex virus in liver

1. Histological Stains
Cytomegalovirus in alveolar cells

Herpes virus

Electron Microscopy
Rota virus

Detect virus products

Respiratory syncitial virus infected cell

Antigens

Amplification of

Nucleic acids

viral nucleic acid PCR

Grow viruses
ISOLATION OF VIRUSES 1.Tissue culture 2. Animal inoculation

1. Cytopathic effect (CPE) 2. Virus neutralisation 3. Haemadsorption

1. Characteristic disease 2. Death & PM findings

Inoculation of embryonated eggs

Tissue culture

Monolayer of cells (human fibroblasts)

Cytopathic effect (CPE)

Diagnosis of viral diseases

1. Identification of Virus
- See virus/virus mediated changes - Detect antigen or nucleic acid of virus - Grow virus

2. Identify host response to virus - Virus specific antibody detection

Immune System : 2 arms

The innate immune system
a general response to anything other than recognized self cells

The adaptive immune system

a specific counter-assault against a known foreign invader

The adaptive immune system

Antigen presentation

T and B cell involvement

Effector Mechanisms
IgM (Acute) X
IgM for hepatitis A virus IgM for dengue virus

IgG (Convalescent) >4X With time total antibody titre rises

Detect host response

Antibody detection
IgM High IgG

Single titre

Acute N

1/5

1/10

1/20

1/40

1/80

Rise in titre

N Convalescence

1/5

1/10

1/20

1/40

1/80

Common assays used in viral diagnosis

Immunoassays
Enzyme linked immunosorbent assay (ELISA) Immunofluorescence assay (IFA) Rapid immunoassays Immunohistochemistry

Molecualr assays
Polymerase chain reaction (PCR) Quantitative polymerase chain reaction (qPCR)

Diagnosis of virus disease in the laboratory can be done by

1.Culturing the virus on enriched blood agar
2. Demonstrating virus inclusion bodies by light microscopy 3. Demonstrating virus particles by light microscopy 4. Demonstrating virus specific IgM antibody response to the virus 5. Demonstrating a four fold rise in antibody titre in convalescent sera