Commonly Used Methods For Individual Viruses  

Virological Methods Slideset

A. DNA Viruses

1. HSV

  1. Virus isolation - HSV is among the easiest viruses to cultivate and it usually only takes 2 to 3 days for a characteristic CPE to appear. A variety of cell lines can be used includinf Vero, Hep-2, human diploid, rabbit kidney, human amnion, rd. Typed by IF (monoclonal Abs), neutralization, REA.

  2. Direct IF of vesicular cell smears

  3. PCR on clinical specimens

  4. EM of vesicle cell smears - cannot be distinguished from VZV particles.

  5. Serology - primary infection may be diagnosed by rising antibody titre or the detection of IgM. Recurrent infection may not be associated with rising antibody titre or an IgM response. Methods available include CFT, neutralization, IAHA, IF, ELISA and RIA.  

2. VZV

  1. Virus isolation - human diploid (HEL, HEK), typical CPE. Virus is difficult to isolate because it is quite labile and vesicle fluid must be inoculated as soon as possible after collection

  2. Direct IF of vesicular cell smears

  3. PCR on clinical specimens

  4. EM of vesicle cell smears

  5. Serology - CFT can be used for diagnosis but not sensitive enough for immunity screening. IF and ELISA may be used for immune status screening  

3. CMV

  1. Virus isolation - HEL (human embryonic lung fibroblasts e.g. MRC-5 cell line) used, takes 2 to 4 weeks for a characteritic CPE to appear. Rapid culture methods such as the DEAFF test may be used that yield a positive result within 48 hours.

  2. CMV antigenaemia test - the expression of the pp65 CMV antigen among neutrophils is detected. A quantitative result (no. of positive cells) is available that is roughly predictive of the severity of the infection.

  3. PCR for CMV-DNA - there is a trend towards the use of quantitative PCR assays.

  4. Serology - CFT widely used but may give false negative result because of low sensitivity. ELISA and latex agglutination are more sensitive serological tests and may be used for immune status screening.  

4. EBV

  1. Virus isolation - presence of EBV can be demonstrated by the transformation of fetal lymphocytes and establishment of permanent lymphoblastoid cell lines expressing EBV antigens.

  2. Molecular techniques such as hybridization and PCR may be used to demonstrate the presence of EBV genomes in biopsy specimens.

  3. Serology - antibodies against VCA, EA, and EBNA may be tested for. P3HR-1 or B-95 cell lines are used for VCA antibody testing. Raji cell line is used for EBNA, testing for EA is carried out on non-producer cell lines induced for the expression of EA. Indirect IF is used for VCA Ab testing, and anticomplement indirect IF must be used for EBNA Ab testing.

From the profile of anti-VCA and anti-EBNA

  1. Susceptible if anti-VCA is absent

  2. Primary infection if only anti-VCA is present. Anti-VCA IgM may be used to confirm primary infection. Rising titres of IgG are not usually seen.

  3. Immune with past infection if both anti-VCA and anti-EBNA are present

Anti-VCA IgA is useful for the screening for cases of NPC and possibly for monitoring of therapy against the cancer.

5. Adenoviruses

  1. Virus isolation - Hep2, HEK. CPE in 2 to 7 days, positive identification by IF, typed by neutralization

  2. IF of NPA specimens

  3. PCR on NPA and other respiratory specimens

  4. Serology - CFT is test of choice, however young children often do not respond with CF antibody after infection. HAI with monkey (Group I), or rat (Groups 2 and 3) erythrocytes may be used. Micro-neutralization tests are the most sensitive and type-specific tests available.  

6. Papillomaviruses

Human papillomaviruses may be detected by EM, IF, hybridization, and PCR techniques on swabs, scrapings or biopsies of lesions. Serology has no role in the diagnosis of HPV infection because the Ab response may require several months to form and Ab levels are usually low.  

7. Polyomaviruses

  1. Virus isolation - JCV requires primary fetal glial cells, BKV will grow in HEK or human diploid fibroblast cells.

  2. Diagnosis of PML may be made by cytological studies of the lesions, the demonstration of JCV antigen in the lesions and demonstration of JCV DNA.

  3. UTI may be diagnosed by cytomorphological appearance of the urinary tract epithelial cells.

  4. Serology is of little value as most people are infected. However a negative result would be strong evidence against a diagnosis of PML.    


B. RNA_Viruses

1. Enteroviruses

  1. Virus isolation

  1. Poliovirus - readily isolated in a large range of cell lines e. g. MK, BGM, LLC-MK2, human diploid, Vero, Hep-2

  2. Coxsackie A - some will grow in Rhadomyosarcoma (Rd) cell lines. Otherwise will require inoculation into newborn mice

  3. Coxsackie B - MK, BGM, LLC-MK2, vero, hep-2

  4. Echovirus - MK, BGM, LLC-MK2, human diploid, Rd

The isolate may be identified by neutralization test using serum pools such as the Lim Benyesh-Melnick pool (A-H pool, will identify 42 enterovirus serotypes). If a single identification cannot be made, then the isolate may be a mixture.

  1. Serology - neutralization tests are the most sensitive and specific tests. Metabolic inhibition test (colour test) is based in the same principle and employs known quantities of cell suspension that are added to test tubes 1 hour after the serum-virus mixture. Continued growth of cells will lower the pH of the medium which will be indicated by a colour change. Another form of the neutralization test is the plaque reduction test. Other serological tests include the CFT, HAI, EIA and RIAs. The CFT is of limited value in terms of specificity because of major cross-reactions. HAI is limited by the fact that only a third of known enteroviruses haemagglutinate.

  1. Rapid detection of enterovirus by PCR has become more widely used especially for the diagnosis of enterovirus meningitis using CSF specimens

2. Influenzaviruses

  1. Virus isolation - MK, LLC-MK2, MDCK. Influenza B will produce a CPE in MDCK cells. Otherwise, the presence of the virus is detected by haemadsorption. Further identification is made by IF and HAI tests

  2. Detection of virus antigen by IF or EIA

  3. RT-PCR on respiratory specimens

  4. Serology - CF test can be used for new virus subtypes, whereas HAI is subtype-specific

3. Parainfluenza_viruses

  1. Virus isolation - MK, LLC-MK2

  2. Direct detection of virus antigen by IF

  3. RT-PCR on respiratory specimens

  4. Serology - CFT, young children do not develop CF antibody well on initial infection. Also, some degree of cross-reaction is present. HAI tests are more type-specific than the CF test but cross- reactions are still present. Neutralization (haemadsorption- inhibition) tests carry the greatest specificity. EIAs are available for serological diagnosis

4. Mumps

  1. Virus isolation - Mumps virus may be isolated from the urine, saliva or CSF specimens. MK, LLC-MK2, HEK, Vero. The CPE consists of syncytial formation. Haemadsorption should be carried out since the CPE does not always occur on the first passage. Confirmation by IF or haemadsorption-inhibition

  2. RT-PCR on CSF and other specimens

  3. Serodiagnosis - CFT is widely used for serological diagnosis. Antibodies against the S antigen appears within a few days after the onset of symptoms. Antibodies to the V antigen seldom appears within 2 weeks of the onset of symptoms but are present at 4 weeks. Antibodies against the S antigen are transient in comparison to those against the V antigen. Other methods available include HAI, SRH, IF and EIAs.    

5. Measles

  1. Virus isolation - MK, HEK cells may be used. CPE takes a week or more to appear. The cell cultures may be examined by haemadsorption or by IF. CPE consists of multinucleated giant cells. Definitive identification requires neutralization. Measles may be isolated from throat swabs, conjunctival swabs, and urine. Recovery of measles from SSPE specimens require special processing and cocultivation techniques.

  2. Direct detection - typical multinucleated giant cells may be found by direct examination of nasopharyngeal secretion or conjunctival swab. IF may be used to stain buccal, nasopharyngeal, or urinary sediment smears.

  3. RT-PCR on clinical specimens

  4. Serology - CFT and HAI are widely used. CF is less specific than HAI. The CF antibody rises and peaks later than the HAI antibody.

5. RSV

  1. Virus isolation - Hep-2 cells are the most sensitive. A characteristic CPE is produced. If CPE is not present, IF may be carried out

  2. Direct detection - IF or ELISA may be carried out on NPA

  3. RT-PCR on NPA

  4. Serological diagnosis - CFT and ELISA may be used. It is not unusual to find antibodies in acute phase sera. In the very young, these represent transplacental antibodies, in older children, they are the result of previous RSV infection.  

6. Rabies

  1. Virus isolation - intracerebral inoculation of laboratory mice is the method of choice. Rabies virus will propagate in a variety of cell cultures including human diploid cells but have not replaced the mouse system

  2. Direct detection - specimens are examined for the presence of Negri bodies and rabies antigen by IF. Neck skin biopsy and corneal smears are the specimens of choice. Neck skin biopsy is more sensitive than corneal scrappings.

  3. RT-PCR on skin biopsies and saliva

  4. Serological diagnosis - the mouse neutralization test is the standard method. ELISAs are also available. Serum or CSF specimens may be used.  

7. Arboviruses

  1. Virus isolation - the arbovirus is usually present in the blood during the acute febrile phase of the illness. To reduce the effect of serum inhibitors, isolation attempts should be made not only with undiluted serum, but dilutions of 1:5 or 1:10 or higher. Virus isolation is not usually attempted from tissue biopsies with the exception of brain tissue from patients with encephalitis. 3 systems are available for virus isolation;-

  1. intracerebral inoculation of newborn mice

  2. tissue culture eg. LLC-MK2, Vero, artrhopod cell lines

  3. intrathoracic inoculation of arthropods

Identification is carried out using serological techniques with reference typing antisera. IF, HI or EIA may be used.

  1. Direct examination - IF may be used to demonstrate arbovirus antigen in tissues

  2. RT-PCR on clinical specimens

  3. Serology - CFT and HAI are the most commonly used tests for detecting arbovirus antibodies. Other tests which can be used include IF, RIA, EIA, and neutralization test.

8. Arenaviruses

  1. Virus isolation - virus may be isolated from the blood during the acute febrile phase of the illness. The standard procedure involves the inoculation of Vero cells followed by IF

  2. Direct detection - these methods are possible but not routinely used because of the need for category 4 containment facilities

  3. Serological diagnosis - IF, CFT, neutralization and ELISA may be used.  

Virological Methods Slideset