Laboratory Diagnosis of Viral Infection

In this article we will discuss about the Laboratory Diagnosis of Viral Infection.

Introduction:

The choice of materials and methods for laboratory confirmation of viral infection depends on the stage of illness (Table 13.1).

The following methods are commonly employed:

(a) Microscopy: Cytopathic effect, inclusion bodies.

(b) Culture and isolation: Laboratory animals, chick embryo, tissue culture, cell culture.

(c) Serology: HI, CFT, EIA, Western blot test.

(d) Detection of viral proteins and genetic material: DNA probes, PCR.

Specimens:

Specimens to be collected early in the acute phase of the disease before the virus ceases to shed (Table 13.2).

Collection and transport of specimens:

Ideally all specimens for detection of virus should be processed by the laboratory immediately because many viruses are labile and the samples are also susceptible to bacterial and fungal overgrowth. Specimen should be placed in ice and transported in special media (Stuart’s viral transport media) containing proteins.

Blood for serological test is transported to laboratory in sterile test tube. Serum is separated as soon as possible. Blood for viral culture is to be transported in a sterile vial containing anticoa­gulant. Blood should be refrigerated at 4°C until processing, and can be stored for months at -20°C or below. Virus specific IgM should be tested before freezing since IgM may form insoluble aggregates upon thawing.

A. Microscopy (cytology):

Viruses produce cytopathic effect (CPE) which include: change in cell morphology, cell lysis, vacuolation, syncytia formation and inclusion bodies (Table 13.3).

1. Cytopathic effect (CPE):

Viruses cause cell degeneration or cell death which can be seen by microscopical examination of cultures. Each infectious virus particle gives rise to a localised focus of infected cells (a focus of cytopathology) that can be seen with the naked eye (Fig. 13.5). Such foci are called plaques and each plaque represents an infectious virus. Cell degeneration is manifested by certain changes.

(a) Syncytium formation (Fig. 13.1):

Syncytium or multinucleated giant cells result from fusion of con­tiguous cells in the monolayer as seen in measles virus.

(b) Cell necrosis and lysis:

Infected cells become pyknotic and granular, e.g. entero-viruses.

(c) Cellular clumping:

The cells do not fuse but produce large clumps resembling clusters of grapes, e.g. adenovirus.

(d) Inclusion bodies:

Inclusion bodies are virus- specific intracellular globular masses produced during replication of virus in host cells and visible under the light microscope. They are far larger than a single mature virus particle (elementary body), size ranges from 20-25 μm and can be seen by light microscope.

Inclusion bodies formed by different viruses show distinct shape, size, location and staining properties and their presence in an infected cell is a presumptive histological evidence of viral infection.

These are generally acidophilic (eosino­philic) structures, appear pink when stained by Giemsa’s or eosin-methylene blue stain and exa­mined under light microscope. Some viruses produce basophilic inclusions (adenovirus). They may appear round, globular, oval or irregular.

Site of occurrence:

(i) Intracytoplasmic Inclusion bodies may be formed in the nucleus, cytoplasm or both (Figs. 13.2 & 13.3):

(i) Intracytoplasmic bodies:

Small pox (Guarnieri bodies), rabies (Negri bodies), yellow fever (Councilman bodies). Inclusion bodies produced in fowl-pox virus infections are called Bollinger bodies which are larger than that formed in vaccinia virus infections. Intracytoplasmic molluscum bodies (20-30 μm) are large.

(ii) Intra-nuclear bodies:

These were classified by Cowdry (1934) into two types: Cowdry A in­cludes inclusion bodies of variable size and of granular appearance (herpes virus, yellow fe­ver virus) and type B of more circumscribed inclusion (adenovirus, poliovirus). Large intra­-nuclear inclusion is also seen in CMV infection of cell.

(iii) Inclusions formed in both nucleus and cytoplasm:

Measles virus infection.

(e) Discrete focal degeneration:

It is found in her­pes virus infections.

(f) Transformation:

Oncogenic virus transforms cells and causes loss of contact inhibition and the cell growth appears in a piled-up fashion producing “micro-tumours” (Fig. 13.1).

2. Immuno-electron microscopy:

When virus- specific antibody is added to a sample, the virus particles clump and thereby facilitate the detection and simultaneous identification of virus. This technique is useful in diagnosis of enteric and rota viruses which are found in abundance and have a characteristic morphology.

B. Culture and isolation:

Culture and isolation is the gold standard for prov­ing a viral aetiology of a syndrome (Table 13.4):

1. Laboratory animals:

In the past, white mice and chimpanzees were inoculated with specimen for viral cultivation, but inoculation of laboratory animals has been largely replaced by the use of cell cultures.

Suckling mice (less than 48 hours old) are very susceptible to toga and coxsackie viruses, which are inoculated by intra-cerebral or intranasal routes.

2. Chick embryo (embryonated egg):

Eggs are kept in the incubator and embryos 7 to 12 days old are used. The embryonated egg is in­oculated by one of the several routes (Fig. 13.4).

After inoculation of the chick embryo, it is incubated and examined daily for virus growth:

(a) Chorioallantoic membrane (CAM):

CAM is inoculated mainly for growing poxvirus. Viral replication produces visible lesions (pocks), gray-white area in the transparent CAM. Each pock is derived from a single virion. Pocks produced by different viruses have different morphology.

(b) Allantoic cavity:

Allantoic inoculation is done mainly for production of vaccine of influenza virus. Other allantoic vaccines include yellow fever (17D strain) and rabies (flury strain) vaccines. Duck’s egg, being larger, provide better yield of rabies virus than hen’s egg.

(c) Amniotic sac:

It is mainly inoculated for pri­mary isolation of influenza virus.

(iv) Yolk sac inoculation:

It is inoculated for cul­tivation of some viruses as well for some bac­teria (chlamydiae and rickettsiae).

3. Tissue culture:

There are three types of tissue culture — organ cul­ture, explant culture and cell culture. Small bits of organs (organ culture) from man and animal are maintained in tissue culture growth medium. Organ cultures are done mainly for highly specialised parasites of certain organ, e.g. tracheal ring culture for isolation of corona virus. Explant culture is rarely done nowadays.

The cell culture method is routinely employed nowadays for identification and cultivation of viruses. The growth medium for tissue culture is basically a balanced salt solution and contains 13 essential amino acids, glucose, salts, buffering system, protein supplement (lactalbumin hydro- lysate), calf serum (5%), antibiotics (penicillin, streptomycin) and phenol red (indicator).

Cell Culture:

Tissue fragments are trypsinised and the dissoci­ated cells are washed, counted and suspended in a growth medium. Most of the cell types undergo a very slow cell division once in 24 to 48 hours. Cell suspension is distributed in tubes, bottles or Petri dishes. Cells of fibroblastic or epithelial nature adhere and grow in the glass surface.

On incubation, the cells divide and spread out on the glass surface (wall of test tube) to form a confluent monolayer sheet of cells within a period of one week:

(a) Types of cell cultures:

Cell culture are classified into three types (primary cultures, diploid cultures, heteroploid or continuous cell line cultures) on the basis of their origin, chromo­somal characters and the number of genera­tions through which they can be maintained (Table 13.5).

(b) Permissiveness:

Since all viruses do not grow in all cell lines, as a virus does not grow in a cell unless it carries receptor for the virus. Hence, specimen is to be inoculated in 3 to 4 cell lines with the hope that at least one of them will be permissive for the unknown virus.

(i) Primary cell culture:

These are normal cells obtained from fresh organs of animal or human being and cultured. Once the cells get attached to the vessel surface, they undergo mitosis until a confluent monolayer of cells covers the surface.

These cells are capable of only limited growth in culture and cannot be maintained in serial culture. They are commonly employed for primary isolation of viruses and in pre­paration of vaccine. The examples of primary culture include monkey kidney cell (Fig. 13.5), and human amnion cell culture.

(ii) Diploid cell cultures (Semi-continuous cell lines):

These cells of a single type, usually fibroblasts, contain the same number of chro­mosomes as the parent cells and are diploid. The diploid cell strains can be sub-cultured for lim­ited number of times. There is a rapid growth rate and after about 50 serial subcultures they undergo “senescence” and the cell strain is lost.

The diploid cell strains are susceptible to a wide range of human viruses. They are also used for isolation of some fastidious viruses and production of viral vaccines. The fibro­blasts are usually derived from embryo tissues (human embryo lung strains).

(iii) Heteroploid cultures (Continuous tumour cell lines) (Fig. 13.6):

These are cells of a single type capable of infinite growth in vitro. They are derived from immortalized cell lines (cancer cells), often of epithelial origin. These cells grow faster and their chromosomes are haploid. They are termed continuous cell lines as they can be serially cultivated indefinitely.

The standard continuous cell lines have been derived from human cancers, such as HeLa (derived from cervical cancer of a lady, Hela by name), HEp2 and KB cells.

Continuous cell lines are maintained either by serial subculture or by storing in deep freeze at -70°C so that these can be used when necessary. These are not used for preparation of viral vaccines, as vaccines prepared in cancer cells is considered unsafe for human use.

Primary cell cultures are generally best for virus isolation and rhesus monkey kidney cell cultures are widely used, which are sensitive to a wide range of viruses.

Identification:

For precise identification of a vi­rus grown in cell cultures, several additional tests have to be performed:

1. Morphologic examination:

Morphological study of a virus can only be made by electron microscopy which is technically difficult and expensive. Moreover, this may not lead to success­ful identification of the virus.

2. Serological test:

Serologic tests help in most cases for precise identification of the virus:

(i) Neutralisation:

It can be performed with any virus producing CPE. Standard known antiviral serum is mixed with the unknown virus recovered from the cell culture medium.

After incubating the antibody-virus mixture, it is inoculated into a fresh cell line. Specific antiserum neutralizes virus activity during incubation, and, consequently, the mixture fails to produce specific CPE. The haemadsorption viruses are typed by neutralisation test.

(ii) Haemadsorption:

Cells infected with influenza virus, para-influenza virus, mumps virus, and toga virus express a viral glycoprotein (haemagglutinin) in the cell membrane that binds or adsorbs red blood cells of defined animal species to the infected cell surface (called haemadsorption) (Fig. 13.7).

(iii) Haemagglutination:

When the above-mentioned viruses-orthomyxo, paramyxo and toga viruses), carrying haemagglutinin in their envelope are shed into the culture medium, they can be detected by the agglutination of erythrocytes, a process called haemagglutination.

(iv) Haemadsorption inhibition test:

This is observed with haem agglutinating viruses (orthomyxo; paramyxo and toga viruses). Medium from the cell culture is mixed with a specific antiserum, and the mixture is added to a fresh culture. After incubation, the second culture is tested for haemadsorption. A negative result indicates that the antiserum is specific and has reacted with the virus and, thereby, blocking haemad­sorption.

(v) Cytopathogenic effects interference inhibition test:

Specific antibody to the unknown interfering virus (e.g. rubella virus, when it is the suspe­cted identity of the unknown virus) is added to medium obtained from the cell culture. The mixture is then inoculated into a second culture.

After incubation for 2-3 days, the cytopathic virus (e.g. echovirus) is added to the second cell culture and incubated for 1-2 days. If cytopathogenic effects have occurred, it is concluded that the first culture was infected with the interfering virus against which the antiserum was employed.

(vi) Direct immunofluorescence:

A cell culture is inoculated with a specimen and incubated. Af­ter 24—48 hours the virus can be identified by adding a fluorescent monoclonal antibody to the suspected virus to a scraping obtained from the infected culture.

The scraping is then incubated for a short time, followed by washing to eliminate unreacted antiserum, the scraping is examined — under the fluorescent microscope. When the cells fluoresce, the vi­rus in question is responsible for the infection of the monolayer.

(vii) Immuno electron microscopy (IEM):

Although a virus can be visualised by electron microscope, but precise identification of a virus (e.g. herpes simplex, VZ, CMV and Epstein—Barr) is not possible on a mere morphological basis. IEM uses a specific antibody labelled with electron- dense tag to specifically identify the virus. The technique is very expensive and not useful for routine purpose.

C. Serology:

Serology is useful and simpler in diagnosis in many cases. The course of the infection (acute or chronic) can also be determined by serology.

1. General considerations:

Total antibody assays are done, usually to ob­serve a significant rise of specific antibodies to a suspected virus on two paired samples, first when the patient is ill and second during convalescence (2 to 3 weeks later). There is usually a four-fold rise in antibody titre. The diagnosis is usually retrospec­tive and mainly useful in epidemiological study.

2. Techniques:

(a) Haemagglutination inhibition (HI) is used for the measurement of antibodies directed against haem agglutinating viruses, e.g. influenza viruses. Antibodies to the viral haemagglutinins in serum prevents a standardised amount of virus binding to and agglutinating erythrocytes.

(b)Complement fixation test (CFT) is the main­stay for some viral diseases. However, it can only detect CF-antibodies (i.e. IgG and IgM) and cannot differentiate between the two.

(c) Direct immunofluorescence test:

A fluorescein —Conjugated antiserum of the suspected virus is mixed with the infected tissue sample and examined under a fluorescent microscope for pale green fluorescent areas on the slide.

(d) Enzyme immunoassay (ELA):

(i) Measurement of antibody by EIA:

A known antigen is adsorbed to a solid phase-like test tube, micro-titre plate well to which proper dilution of patient’s serum is added and then incubated. If there is antibody in patient’s serum, it will attach to the antigen. The solid phase is washed off the un-reacted immunoglobulin’s.

After washing enzyme- labelled anti-human immunoglobulin (AHIG) is added to the solid phase. The solid phase is again washed off to remove unreacted antibody (AHIG). Then a subst­rate is added to the solid phase. The enzyme-labelled antibody (AHIG) binds to the patient’s antibody that has reacted with the immobilised antigen.

The enzyme splits the substrate to a colour com­pound. The intensity of the colour is proportional to the quantity of antibody present in the patient’s serum. The colour is measured by ELISA reader. These tests are rapid (takes 10-15 minutes) and easy.

(ii) Measurement of antigen:

A known specific antibody is adsorbed to the solid phase (e.g. well in micro-litre plate). Patient’s serum is added, and if viral particles or viral antigens are present in the circulation, they will be bound by the immobilised antibody. The solid phase is washed.

Then enzyme-la- belled antibody (e.g. AHG) of the same specificity to the solid phase is added. Af­ter incubating and washing off unbound reactants, a chromogenic substrate is added. A positive reaction is indicated by the de­velopment of colour.

(e) Western blot (immunoblot) test:

It is a most widely used confirmatory test for detection of positive HIV antibody immunoassay.

The test is done as follows:

(i) HIV antigens are separated by electro­phoresis on polyacrylamide gel which is blotted onto nitrocellulose paper strips.

(ii) The strip is incubated with patient’s anti­body.

(iii) After washing to remove the unbound antibody, an enzyme labelled anti-human globulin (AHG) antiserum is placed on the strip. The labelled antibody binds to any antibody captured by the viral antigens. When a chromogenic substrate is added subsequently, the antigen-antibody com­plexes are revealed as stained bands which represent the major antigenic proteins of HIV.

D. Detection of viral genetic material:

(1) Nucleic acid probes:

Nucleic acid probes are short segments of DNA complementary to specific regions of a viral genome. DNA probe analysis is especially useful to detect slowly replicating or non-productive viruses, e.g. CMV, human papilloma viruses and HIV (during window period).

(2) Polymerase chain reaction (PCR) and reverse transcriptase PCR:

PCR is a method that duplicates short DNA segments thousand to a million fold. When the viruses are present in low concentrations, they can be duplicated (amplified) using PCR.

Viral Assays:

1. Total particle count:

Total count of viruses irrespective of whether they are viable or nonviable is estimated by electron microscopy and haemagglutination:

(a) Electron microscopy:

The virus particles in a negatively stained suspension can be counted directly. The commonest method is to mix virus suspension with known concentration of latex particles and studied under the electron microscope. The number of virus particles in the suspension are calculated from the ratio of latex spheres to virus particles.

(b) Haemagglutination (HA):

Although it is not a very sensitive method, yet HA titre gives an approximate count. About 107 influenza viruses are necessary to bind erythrocytes (0.5 ml of 0.5% suspension) into a lattice-like aggregates of macroscopic size.

2. Infectious virions assay (assay of infectivity):

Quantitative infectivity assay:

The quantitative infectivity assay measures the number of viable infectious particles in a suspen­sion.

This can be measured by two methods:

(i) Plaque assay:

Serial dilutions of a virus sus­pension are inoculated into confluent mono­layers of culture cells in a Petri dish or bottle. After allowing time for adsorption (1 to 4 hours), the monolayer is covered with agar gel which prevents diffuse spread of the viruses readily through the fluid medium.

Moreover, the agar gel layer restricts spread of the released progeny particles in such a way that only neighbouring cells are infected. Each infectious particle gives rise to a localised focus of infected cells, called a plaque, which can be seen with the naked eye (Fig. 13.5).

The titre of viruses is expressed in plaque forming units (PFU) per volume. Since each plaque represents an infectious virus, the plaque titre is the infectivity titre.

(ii) Pock assays:

Since each pock in CAM arises from infection by a single virus particle, the number of pocks formed corresponds with the number of viruses present in the inoculum.

Antiviral Susceptibility Testing (AST):

It is done for defining mechanisms of antiviral resistance and to assess the frequency with which drug-resistant viral mutants emerge in clinical practice.

There are two types of AST:

(i) Phenotypic assays:

These are in vitro suscepti­bility tests. Cell culture is incubated with several dilutions of the drug to which a constant amount of virus has been added. The test measures the reduction in the number of plaques, inhibition of viral DNA synthesis, and reduction in the yield of viral structural proteins (e.g. gp 24 of HIV).

(ii) Genotypic assays:

These analyse viral nucleic acids to detect specific mutations respon­sible for drug resistance. CMV shows UL 97 (phosphotransferase) and UL 54 (polymerase) mutants with ganciclovir resistance. These are detected by PCR amplification and sequencing of entire UL 54 gene and fragment of UL 97 gene.