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.
Direct IF of vesicular cell smears
EM of vesicle cell smears - cannot be distinguished from
VZV particles.
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
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
Direct IF of vesicular cell smears
EM of vesicle cell smears
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
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.
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.
PCR for CMV-DNA - there is a trend towards the use of
quantitative PCR assays.
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
Virus isolation - presence of EBV can be demonstrated by
the transformation of fetal lymphocytes and establishment
of permanent lymphoblastoid cell lines expressing EBV
antigens.
Molecular techniques such as hybridization and PCR may be
used to demonstrate the presence of EBV genomes in biopsy
specimens.
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
Susceptible if anti-VCA is absent
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.
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
Virus isolation - Hep2, HEK. CPE in 2 to 7 days, positive
identification by IF, typed by neutralization
IF of NPA specimens
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
Virus isolation - JCV requires primary fetal glial cells,
BKV will grow in HEK or human diploid fibroblast cells.
Diagnosis of PML may be made by cytological studies of
the lesions, the demonstration of JCV antigen in the
lesions, demonstration of JCV DNA.
UTI may be diagnosed by cytomorphological appearance of
the urinary tract epithelial cells.
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
Virus isolation
Poliovirus - readily isolated in a large range of
cell lines e. g. MK, BGM, LLC-MK2, human diploid,
Vero, Hep-2
Coxsackie A - some will grow in Rhadomyosarcoma (Rd)
cell lines. Otherwise will require innoculation into
newborn mice
Coxsackie B - MK, BGM, LLC-MK2, vero, hep-2
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.
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.
Rapid detection of enterovirus by PCR has become more
widely used especially for the diagnosis of enterovirus
meningitis using CSF specimens
2. Influenzaviruses
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
Detection of virus antigen by IF or EIA
Serology - CF test can be used for new virus subtypes,
whereas HAI is subtype-specific
3. Parainfluenza_viruses
Virus isolation - MK, LLC-MK2
Direct detection of virus antigen by IF
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
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
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
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.
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.
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
Virus isolation - Hep-2 cells are the most sensitive. A
characteristic CPE is produced. If CPE is not present, IF
may be carried out
Direct detection - IF or ELISA may be carried out on NPA
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
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
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.
Serological diagnosis - the mouse neutralization test is
the standard method. ELISAs are also available. Serum or
CSF specimens may be used.
7. Arboviruses
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;-
Identification is carried out using serological techniques
with reference typing antisera. IF, HI or EIA may be used.
Direct examination - IF may be used to demonstrate
arbovirus antigen in tissues
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
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
Direct detection - these methods are possible but not
routinely used because of the need for category 4
containment facilities
Serological diagnosis - IF, CFT,
neutralization and ELISA may be used.
