LAB DIAGNOSIS OF VIRAL INFECTIONS Electron Microscopy viruses

LAB DIAGNOSIS OF VIRAL INFECTIONS

Electron Microscopy • viruses growing and multiplying in cell cultures can be detected using electron microscope. • Viruses may be detected in the following specimens. • Feces - Rotavirus, Adenovirus, Norwalk like viruses, Astrovirus, Calicivirus • Vesicle Fluid – HSV, VZV • Skin scrapings - papillomavirus, orf, molluscum contagiosum

Electronmicrographs Adenovirus Rotavirus

Immune Electron Microscopy • The sensitivity and specificity of EM may be enhanced by immune electron microscopy. • There are two variants: • Classical Immune electron microscopy (IEM) • Solid phase immune electron microscopy (SPIEM)

Cultivation and detection of viral growth • Three methods are employed for cultivation of viruses. • Animal Inoculation. • Embryonated egg Inoculation. • Tissue Culture.

Animal Inoculation • is used for primary isolation of certain viruses, to study pathogenesis of viral diseases and to study viral oncogenesis. • The animal used are • Infant (suckling) mice – for isolation of Arboviruses and Coxsackieviruses. • Routes of inoculation include intracerebral, intraperitoneal, intranasal and subcutaneous. • After inoculation, animals are observed for signs of disease or death.

• Ferrets – for isolation of influenza viruses by intranasal inoculation. • African Green Monkeys – for studying HIV virus.

Embryonated Egg Inoculation • Embryonated hens eggs (7 – 12 days old) are inoculated by several routes such as – • Chorioallantoic Membrane (CAM) – used for growing mainly pox viruses and herpes viruses. • It produces visible lesions called pocks. Pocks produced by different viruses have different morphology.

• Amniotic Cavity – used for primary isolation of influenza virus. • Allantoic Cavity – used for growing influenza virus for vaccine production. • Yolk Sac – used for cultivation of certain Arboviruses and bacteria like Chlamydia and Rickettsiae.

Embryonated Eggs

Foci formation on the chorioalantoic membrane by vaccinia virus

Small Pox Vaccine Infected Chorioalantoic Membrane

Tissue Culture • Organ Culture – small bits of organs maintained in the tissue culture growth medium can be used for isolation of certain viruses. E. g. , tracheal ring culture for isolation of Coronaviruses. • Explant Culture – fragments of minced tissue can be grown as explants. This method is rarely used nowadays.

Cell Culture • Tissues are dissociated into component cells by the action of Proteolytic enzymes such as trypsin. • The cell suspension is distributed in glass or plastic tubes or Petri dishes. • On incubation, the cells divide to form a monolayer sheet of cells within a period of one week.

• Viruses can be grown in living cells maintained in sterile conditions in the laboratory. • There are three types of tissue cultures.

Primary Cell cultures • These are normal cells, freshly taken from the organs of animal or human beings and cultured. • They are capable of only 5 – 10 divisions and employed for primary isolation of viruses and vaccine production. • E. g. – monkey kidney cell; human amnion cell; chick embryo fibroblast cell cultures.

Diploid cell Cultures • These are cells of a single type that contain same number of chromosomes as the parent cells and are diploid. • They can be sub-cultured up to 50 times. E. g. WI 38 (Human Embryonic Lung cell Line).

Continuous Cell Cultures • These are cells of single type that are capable of indefinite growth in vitro. • These are usually derived from cancerous tissue. • They grow faster and have haploid number of chromosomes.

• These cells can be serially cultivated indefinitely and hence, are called as continuous cell lines. . • These can be stored at – 70 C for use whenever necessary. • E. g. He. La cell Lines (Human carcinoma of cervix cell lines); HEp 2 cell lines (Human Epithelioma of larynx cell lines); Vero cell lines (Vervet monkey kidney cell lines).

Detection of viral growth in cell cultures • Virus growth in cell cultures can be detected by the following methods. • Cytopathic Effect (CPE) – many viruses cause morphological changes in the cultured cells. The viruses causing cytopathic effect are called as cytopathogenic viruses. CPE induced by viruses are of the following types –

• Syncytia formation – fusion of infected cells with neighboring cells to form multinucleated giant cells is called Syncytia formation. E. g. Respiratory Syncitial Virus (RSV).

Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom).

• Cell necrosis and lysis – degeneration of entire cell line. E. g. Enteroviruses. • Transformation – infected cells clump together producing microtumours. E. g. Adenoviruses. • Rounding of cells – viral replication leading to nuclear pyknosis, rounding, refractility and degeneration. E. g. Picornaviruses. • Discreet focal degeneration – E. g. Herpes viruses.

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.

ECHO Virus: Round Cells

Round Cells

Foamy Virus Vacuoles

Vaccinia CPE in Monkey Cells

Herpes Simplex Virus Plaque Formation

Haemadsorption • Certain viruses can be detected in cell cultures by addition of guinea pig erythrocytes to cell cultures. • If the viruses are multiplying in the cells, erythrocytes will adsorb to the infected cells. • This is called Haemadsorption. • E. g. Orthomyxo and Paramyxoviruses.

Haemadsorption Syncitial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet.

Immunofluorescence • viruses can be detected in infected cells by staining with fluorescent conjugated antibodies and examined under fluorescent microscope for the presence of viral antigen.

Immunofluorescense Positive Immunofluorescence test for rabies virus antigen.

• Interference – growth of non-cytopathogenic virus can be detected by the subsequent growth of known cytopathogenic virus. Growth of first virus will inhibit the infection by the second virus by interference. E. g. Rubella virus. • Detection of Enzymes – viral enzymes can be detected in cell cultures after the growth of viruses. E. g. Reverse transcriptase enzyme produced by retroviruses.

Rapid Culture Techniques Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. The CMV DEAFF test is the best example, whereby • • • The cell sheet is grown on individual cover slips in a plastic bottle. Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days. The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorscence.

DEAFF test for CMV (Virology Laboratory, Yale-New Haven Hospital)

Serology • Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of Ig. M in primary infection • Following serological methods can be used • Complement fixation test • Haemagglutination inhibition test • Immunofluorescence • Neutralization test • Radioimmunoassay • ELISA • Latex agglutination test • Western blot assay

Haemagglutination Inhibition Test • Serological diagnosis of influenza • Serum diluted serially (1: 10; 1: 20) • Influenza virus suspension with 4 HA units is added • Fowl red cells are added • Highest dilution of serum inhibiting haemagglutination is its titer (HI titer)

VIRAL HAEMAGGLUTINATION

HEMAGGLUTINATION INHIBITION TEST

Neg. Pos. 1/1024 1/512 1/256 1/128 1/64 1/32 1/16 1/8 1/4 Patient 1/2 Agglutination/Hemagglutination Titer 1 64 2 8 3 512 4 <2 5 32 6 128 7 32 8 4

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement fixation obtained with acute and convalescent phase serum specimens, respectively. (2 -fold serum dilutions were used) The observed 4 -fold increase is significant and indicates recent infection.

Neutralization tests • Neutralizing antibodies react specifically with a virus by attaching to the protein in virus capsid or envelope • Virus looses the ability to cause infection • Neutralization depends on time, temperature & concentration of viruses

Neutralization tests • To measure virus neutralizing antibodie in patient’s serum • Serum( in dilutions) + Viral suspension inoculated into test cell culture • Viral suspension inoculated into control cell culture • Failure to develop CPE if Antibodies present • CPE in control cell culture • Four fold rise in Ab titer is required to establish diagnosis

Types • Nt test in cell culture – Polio, herpes – counting no. of plaques; Plaque reduction assay – TCID 50 – 50% Tissue culture infection dose • Nt test in eggs – Influenza and mumps viruses; pock reduction assay • Nt test in mice – Mice of known uniform susceptibility – Inoculated mice observed for 21 days – Illness and death recorded daily

Enzyme Linked Immunosorbent Assay (ELISA) Uses : 1. Detection and quantitation of antibodies in a wide variety of bacterial, viral and parasitic diseases like TORCH syndrome. 2. Detection and quantitation of antigens for the diagnosis of infectious diseases e. g. rotavirus, HBs. Ag. 3. Detection and quantitation of tumor markers e. g. AFP , CEA.

ELISA for HIV antibody Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Western Blot HIV-1 Western Blot • Lane 1: Positive Control • Lane 2: Negative Control • Sample A: Negative • Sample B: Indeterminate • Sample C: Positive

Rapid Diagnosis Based on the Detection of Viral Antigens Nasopharyngeal Aspirate RSV Influenza A and B Parainfluenza Adenovirus Faeces Rotaviruses Adenoviruses Astrovirus Skin HSV VZV Blood CMV (pp 65 antigenaemia test)

Immunofluorescence test • Direct – Antigen is detected by using a specific monoclonal antibody with Fluorescent dye against the antigen • Indirect – Patient serum is reacted with specific antigen and then Anti-Ig. G antibody with fluorescent dye is used tod detect patient’s antibody.

Direct and indirect immunofluorscence methods for antigen detection

Immunofluorescense Positive immunofluorescence test for rabies virus antigen. (Source: CDC) (Virology Laboratory, Yale-New Haven Hospital)

CMV pp 65 antigenaemia test (Virology Laboratory, Yale-New Haven Hospital)

Molecular Methods • Methods based on the detection of viral genome are also commonly known as molecular methods. • Dot-blot, Southern blot, in-situ hybridization are examples of classical techniques. They depend on the use of specific DNA/RNA probes for hybridization. • Detection of virus genetic material (PCR, RTPCR,