Mendiola, Leomill O.

January 01, 2017

BMLS 3 B Dr. Amelda C. Libres, RMT, DODT

VIROLOGY
Baileys and Scotts Diagnostic Microbiology
CHAPTER 65 SUMMARY

HISTORY
HISTORY ATAT GLANCE
GLANCE

The Dawn of examining viral disease traced back as far as 23 B.C that Code of ancient
Mesopotamia noted the bite of the mad dogs can cause disease in humans. Homer, author of
the Iliad, characterizes Hector as rabid.
Aristotles work (The natural history of man) theorized that madness in dogs that causes them to
become more irritable and all the mammals they bite become diseased.
Influenza Virus was once to be the deadliest virus in history which infects a million people in 1700
at Italy resulting from what they thought as the influence of bad air miasma.

VIRUS OVERVIEW/ GENERAL CHARACTERISTICS

The survival of viral infectious agents depends on their ability to infect and reside in a living
organism.
Virus continue its propagation by the means of humans or other host (plant, animals etc., then
establish a long term relationship with the host.
Virus is a submicroscopic (the smallest of all infectious agent, obligate intracellular parasite.
One those who wants to view virus must use Electron Microscope (100,00x higher) because the
size of it only ranges from 20-300 nm (about the size of Staphylococcus). Largest Virus
Poxvirus; Smallest -- Parvovirus
SOME virus are referred to be as tropism (infects to only specific host, and specific tissue).

VIRUS MODIFICATION
Viruses can modify themselves to infect new host. E.g. Avian virus modify to infect humans as
well as SARS, West Nile, influenza A H5 Virus, H1N1 swine flu.
Viruses modify themselves arising from..
o Antigenic shift (major changes that result in novel antigens)
o Antigenic drift (occur infrequently; minor changes)
The result of modification is termed as Novel virus or completely new one.
Emergence of new viral disease (novel virus) is a challenge nowadays (since lack of vaccine,
antiviral agent at some point).
As an effect, the spread may become pandemic (worldwide epidemic of infectious disease).
o E.g. Spanish flu epidemic where arises from avian origin which was responsible for 50
million death worldwide.
o TODAY, human influenza virus is associated to the assortment of genome from avian
influenza virus
GENERAL CHARACTERISTICS
3 MAIN STRUCTURES
1. Nucleic acid core- either RNA OR DNA.
2. Capsid- surrounds and protects nucleic acid, provides Tropism.
3. Envelope- seen in some large viruses, lipid envelope surrounds the virus. Responsible for
the viral entry to the host.
Viral protein found on the envelope
Neuraminidase -
Hemagglutinin (HA) - Marker for viral culture
Glycoprotein spikes - assist stabilization and attachment to the host cell.
 Enveloped Virus tends to be more susceptible of drying on the outside environment thats why
it is transmitted thru direct contact (sexual intercourse, prenatal transmission, respiratory
route).
Non-enveloped/ Naked virus tends to be more resistant because of their greater stability thats
why they can be transmitted thru fecal-oral route.
Nucleocapsid composed of capsid and Nucleic acid core.
o Capsid is typically composed of repeating structural subunit referred as capsomeres.
Nucleic acid genome- encodes proteins for viral penetration, transmission, and
replication.
Nucleic acid Structure- determines the mechanism of viral replication.
(+) strand DNA OR RNA
(-) strand DNA OR RNA

VIRUS TAXONOMY
Regulated by ICTV International Committee on Taxonomy of Viruses
Viral taxonomy is divided into categories: six orders (-virales), 87 families (name ending in
-viridae), 19 subfamilies (-virinae), 348 genera (-virus), and 2290 species.
But medically important viruses composes of 4 orders, 25 families, 12 subfamilies and 66
genera.
Viral taxonomy incorporates variety of categories
o Host range
o Disease pathology
o Antigenicity
o Viral particle properties (size, envelope, capsid structure, physical properties and configuration)
But for now, for simplicity \_()_/
Viral morphology
Method of replication including if its RNA OR DNA
Presence of Envelope
Note: the method or the means of replication refers to how virus duplicate its viral genome.
Note: Molecular sequencing is limited because the instability of viral genome.

VIRAL REPLICATION
IT is referred as infectious cycle.
1. ATTACHMENT (ADSORPTION)
Recognition of the suitable host cell, binding of glycoprotein spikes and carbohydrate
receptors. (See tropism)
2. PENETRATION
It is when the virus enters the cell *note capsid not still removed*
3 types of mechanisms to internalize the viral genome
Fusion of viral envelope to the cell membrane
Endocytosis
Injection of viral nucleic acid
Additionally, adjacent host cell interlinked by the viral envelope which subsequently form a
multinucleated cells referred to as syncytia.
DIAGNOSTIC Note: detection of syncytia means positive for viral cell culture.
3. UNCOATING
It is when virus has been internalized where the capsid is removed due to the degradation
of its own viral enzymes or host enzymes or by simple dissociation.
4. MACROMOLECULAR SYNTHESIS
Production of nucleic acid (Replication) and protein polymers (transcription).
a. Viral transcription- leads to synthesis of mRNA then encodes the ff.
i. Early proteins nonstructural element such as enzymes
ii. Late proteins- structural components
b. Viral Replication- a replication of nucleic acid
 DIAGNOSTIC NOTE: Detection of Early proteins by the means of immunofluorescent
staining techniques means positive for viral cell culture.

5. VIRAL ASSEMBLY
It is when the structural proteins, genomes, and viral enzymes assembled.
Note: Viral envelope is not made from the virus itself but it came from the Nuclear
endoplasmic Reticulum or cytoplasmic membranes from the host during viral budding (the
final step of viral assembly).
6. RELEASE
Its either of the 2 pathway
a) Virus release from the host cell after cell lysis
b) Virus release from the cell by budding which may not result to rapid cell death.
DIAGNOSTIC NOTE: Detection of virus during hemadsorption test reads a positive if
the RBC adsorb to the infected cell membrane due to its affinity to hemagglutinins which
is present in the host cell.
DIAGNOSTIC NOTE: Detection of cell lysis means positive for viral culture but not in
the case if the virus is release from cell budding.
Note: Infected cell host can generate as much as 100,000 virions; but due to rapid replication,
only 1 % of it are viable. Others may die due to error of mutation or may lead to a new novel
virus.
EPIDEMEOLOGY
Aside from the aforementioned mode of transmission, it may be pass thru blood, tissue
transplants, animal bites, and vertical transmission (mother-child).

PATHOGENESIS
PATHOGENESISAND
ANDSPECTRUM
SPECTRUMOF
OFDISEASE
DISEASES
Upper Respiratory tract are the frequent part of the body which virus start to infect.
Viral Infections may produce (3) characteristic clinical presentations
o Acute viral infection (AVI) -evident sign/symptoms
o Latent infection -no visible sign/symptoms
o Chronic infection - arises from AVI. Low level of virus detectable
Viremia occurs after local viral infection, it is where the virus extends its range to secondary
target tissue which body releases mediators of immunity.
Secondary viremia occur when the virus extends the range to variety of tissues.
Symptomatic stage starts where cell mediated immune mechanism tries to prevent replication
of viruses by lysing the infected cell or also directly against virus (immunopathologic reaction).

CHARACTERISTICS OF SOME VIRUS

Some virus can be latent at some point of time

o Note: DNA containing viruses e.g. herpes remain latent in host tissue at a given time.
o During latent stage, the virus now in the cell then the viral genome is now integrated to
the host cells chromosome but still, no viral replication occurs.
o Some Latent viruses can reactivate silently with no clinical symptoms.
o Reactivation may accompany immunosuppression, resulting of recurrence of disease.
Some are ONCOGENIC (Cancer Causing)
o It is when some virus can cross-reacts with related human tissue that result to damage
to host function or known as Autoimmune Pathogenesis.
o As result, after viral infection is resolved, rare viral infection promotes transformation
and immortalization of host cell which lead to uncontrollable cell proliferation.
o Oncogenic Virus is termed to virus which can cause Cancer.
 SPECIAL NOTE: Some are capable to cross react to CNS due to the host Antibody
Just like measles virus, after subsequent replication of Upper respiratory tract and Viremia, it
will lead to secondary viremia ..
o That includes endothelial cell and skin as a target which leads to rashes.
o Hosts Antibody may cross-react to CNS tissue causing Post-Infectious Encephalitis.
o When the virus gets into brain, it may lead to Subacute Sclerosing Pancephalitis.

LABORATORY
LABORATORYDIAGNOSIS
DIAGNOSIS
The demand of clinical laboratory services has skyrocketed during past 2 decades which allows
the discovery antiviral drugs, availability of reagents including rapid tests and cell culture
procedures as well as PCR. Molecular detection is increasingly used for the detection of viruses.

To determine what viral test to offer, one should be aware of the basic status of the patient, the
availability and cost-effective test result.
Where in Viral laboratory, it is a must to get acquainted to all the test and procedure.
Common Equipment needed in the virology laboratory are; BSC , fluorescence microscopy,
inverted bright field microscope, fridge, roller drum for holding cell culture tubes during
incubation and enzyme/molecular testing instrumentation and many more.
BSC level II are needed for community lab center and for nonretroviral laboratories.
BSC level III are specifically used if the virus suspected including H5N1, SARS, Corona Virus,
Smallpox and including virus that can cause hemorrhagic disease.
SPECIMEN SELECTION AND COLLECTION
Specimen collection depends on.
Specific disease syndrome
Viral etiologies suspected
Time of year (some viral disease may have specific time of year to infect)
Selecting specimen is a bit challenging because virus infect tissue distant from the
inoculation site
o e.g. aseptic meningitis, caused by infection with various types of enterovirus, may be
identified by detecting virus in throat, rectal swab, or cerebrospinal uid (CSF) but
pharyngitis and GI symptoms may not be included in the patient complaints
Suspected viruses quite confusing since other virus can ensue also that kind of symptoms.
o E.g. Testing smears of nasal secretions from an infant using uorescence staining
or enzyme immunoassay to detect RSV does not allow for diagnosis of similar
disease resulting from infection with inuenza virus, parainuenza virus, or
metapneumovirus.
Selection of the appropriate specimen is one of the keys to correct test result.
Selection should be with the proper specimen, sample volume and collection timing
In the requisition form, suspected virus should be written to aware the virologist who
performs the test in order to be notified.
Note: Collection timing must be early as possible since virus may be no longer be present
after 2 days after onset of the symptoms
But still there are factors to be considered like patients immune status or age, the type
of virus, and the amount of systemic involvement, may play a role in the length of time
viral shedding is evident, allowing effective laboratory detection. Certain viruses, such as
West Nile virus, produce a brief, low viremia and undetectable levels at the onset of symptoms.
Containers and swabs can enhance the recovery as long as without toxic agent.
Note: Calcium Alginate is toxic to enveloped virus and interferes PCR, fluorescent-
antibody test
TYPES OF SPECIMEN AND ITS IMPLICATIONS
NASOPHARYNGEAL ASPIRATE AND SWAB
Note: Nasopharyngeal Aspirate is superior to throat and nasopharyngeal swabs.
Used for the detection of RSV, Influenza and Parainfluenza (also Swabs).
It is collected by inserting a swab or flexible shaft through the nostril into nasopharynx.
Or by washing/ rinsing with a bulb of syringe containing 3-7 ml of buffered saline.
Nasal specimen is used for the detection for Rhinovirus.

THROAT
Enterovirus, adenovirus, HSV
Throat specimen uses a swab which passes to the inflamed, vesiculated, purulent areas of
the posterior pharynx.
Note: dont touch the tongue, buccal mucosa, teeth and gums.

BRONCHIAL / BRONCHIOALVEOLAR WASHING

Influenza, Adenovirus or any virus which infect lower respiratory tract.

RECTAL SWABS/ STOOL SPECIMENS

Rotavirus, Enteric Adenovirus, enterovirus
Stool specimen are preferred to rectal swabs in the case of enteric Adenovirus.
Rectal swabs are preferred for enterovirus in patients suspected of enteroviral disease
like aseptic meningitis.
Rectal swab is inserted 3-5 cm from anus to obtain feces.
Note: Stool sample is superior to rectal swab alone.
Note: In infants, 5-10 ml either diarrheal or formed stool is preferred for the detection of
enteric adenovirus and rotavirus.

URINE
Cytomegalovirus, Mumps, Rubella, Measles, Polyomavirus, and Adenovirus
2-3 specimen should be obtain
Clean catched specimen with a 10 ml specimen volume
7.5 % Sodium bicarbonate is added to neutralize Ph.
Centrifugation is necessary.

SKIN/ MUCOUS MEMBRANES LESIONS

Enterovirus , HSV, VZV, (CMV and Pox -rare)
Detected by vesicular lesion. Once the vesicle has ulcerated, detection is more difficult.
DIAGNOSTIC NOTE:
Tzanck smear uses two techniques: If its tuberculin syringe is used, the syringe
aspirates the vesicle then flushed to transport medium with phosphate buffered saline.
Or With the roof of the vesicle folded back, excess uid is carefully removed by dabbing
with sterile gauze. A clean glass microscope slide is pressed against the base of the
ulcer. The slide is lifted, moved slightly, and pressed again. Cells from the base of the
ulcer stick to the slide, making an impression smear of infected and uninfected cells.
Additional smears can be made from other vesicles. The slides are sent to the
laboratory for fixation and staining. As an alternative, vesicle uid and cells scraped
from the base of an unroofed vesicle can be added to 2 to 3 mL of viral transport
medium. Smears can be prepared in the laboratory with cytocentrifugation of uid
medium, or PCR can be performed from the specimen in the viral transport medium.

BLOOD
Primarily for the detection of CMV
HSV, VZV, enterovirus, and adenovirus occasionally encountered.
CMV viremia is associated with peripheral blood leucocytes.
Anticoagulated blood (5-10ml) collected in a whole blood tube is needed for CMV.
 Note: EDTA used for Nucleic acid testing or may be serum only.

BONE MARROW
Bone marrow for virus detection should be added to a sterile tube with anticoagulant.
Same as Blood, EDTA used for Nucleic acid testing.
By the means of aspiration except Parvovirus B19.

TISSUE
CMV, inuenza virus, adenovirus, sin nombre virus, brain (HSV), and GI tract (CMV).
Useful for detection of the viral agent that commonly infects lung.
Fresh specimen is preferred for nucleic acid assay. Formalin fixed or paraffin embedded
tissue may be used after deparaffinization and extraction.

GENITAL SPECIMENS
HSV and human papillomavirus (HPV)
Genital swabs should be used for ulcerations and placed in appropriate viral transport
media.
Cervical specimens may be collected using a swab or brush and placed in viral transport
media.

OTHER STERILE BODY FLUIDS

HSV, VZV, Influenza Virus, CMV.

SERUM FOR ANTIBODY TESTING

Detect antibody for specific virus.
The convalescent specimen is collected a minimum of 2 to 3 weeks after the acute specimen.
Acute specimen should be collected as soon as the appearance of symptoms.
3-5 ml serum is obtained by venipuncture.

Note: Respiratory and oral specimen must be decontaminated first due to contaminated bacteria by
the means of centrifugation. But the problem may lead to reduce the recovery of viral agent in
sample.
Note: Many agents of viral gastroenteritis does not tend to grow in cell culture; PCR / electron
microscopy is necessary.

SPECIMEN
SPECIMENTRANSPORT AND
STORAGE AND STORAGE
TRANSPORT

SPECIMEN
The Viral specimen must be processed immediately.
The specimen should be stored at 4C or lower, room temp or higher is discouraged.
When delay of transport is unavoidable, it should be refrigerated, not frozen until processed.
Every attempt should be made to process the specimen within 12-24 hr. of collection.
Storage: 5 days 4C | >6 days -20 -- -70 C,
Specimens for freezing must be diluted (1:2 to 1:5) or emulsified in viral transport medium.
Note: Significant loss of infectivity may occur during prolonged storage especially labile enveloped
virus
REAGENT/ SWABS
Prepared reagent used for testing must be stored at -70 C for no longer than 2-3 days.
Dacron, Rayon swabs are acceptable. Wooden shaft is discouraged.
Note: Swab w/ specimen should be emulsified in viral transport medium before transport.
Calcium alginate not acceptable for the detection of HSV also for the use of PCR.

TRANSPORT MEDIA
 Transport media are useful for maintaining the stability of the virus since it contains Protein
(serum, albumin, or gelatin) and to prevent the overgrowth of the bacteria and fungi by using
antimicrobials.
If serum is used as a stabilizing agent, fetal calf serum is recommended.(less inhibitor)
Transport media examples: Stuart, Amie, Leibevotiz-Emory, HBSS, Eagles Tissue, and M4 AND M5.
If rectal stool specimen: Modified HBSS, Modified Stuart, and Leibevotiz-Emory w/ antimicrobial.

SPECIAL CASE: Blood culture

Serum should be separated from clot as soon as possible
STORAGE : Hour to day 4 C- | week to month -20 C
Testing for Virus specific IgM should be completed before freezing since it may form insoluble
aggregates upon thawing which leads to false negative result.

SPECIMEN PROCESSING
Processing with other microbiology specimen allows viral culture to be processed 7 days a week.
REQUISITION MUST HAVE THIS FOLLOWING
Name
Demographic Record
Source of the Specimen
Virus suspected
Date and time of specimen collection
EQUIPMENT/ DEVICES MUST BE CONSIDERED
o BSC avoid infectious aerosols
o PPE if you dont know this, I have some bad news for you.
o Slightly capped tube avoid infectious aerosols when vortexing
o Protective shield avoid infectious aerosols, use when pipetting
Open only one patient sample at a time when manipulating different specimens.
Aerosols and micro splashes contribute to cross contamination of cultures, especially during viral
respiratory season when a high percentage of specimens are positive for inuenza virus, RSV,
and other viruses.
The fluid specimens must be centrifuge to break up virus containing cells
The viral transport medium must be also centrifuge to re-suspend the medium.
Glass beads are added to transport medium while centrifugation with to break up clump and/ or
release cell aggregates.
IF TISSUE SOURCE
o Mince or ground it first then centrifuge it around (1000x g for 15 min) and supernatant used
for the inoculum. 200-400 microliter of supernatant is needed for cell culture tube.
o If not sufficient supernatant, dilute it with transport medium to increase volume
o If excess, store at -70 C, the excess may be use if the initial inoculum is contaminated
Contaminated specimen still be processed under this following practice in order to be
recultured.
Adding antibiotic-containing viral transport or...
Filtering the specimen using 0.22 to 0.45 microliter filter

Reculturing procedures after the mentioned technique above

1. Specimen allowed to adsorb in an incubator at 35-37 C for 30-60 mins.
2. Then 1-1.5 ml of maintenance medium is then added.
3. Then the tube returned to incubator: preferably Roller rack in the rotating medium.

NOTE: Reculturing under aforementioned circumstance will likely to make virus less
detectable because of additional contamination.
Blood for viral culture requires special processing to isolate leukocyte followed by inoculation into
cell culture tubes.
 DIAGNOSTIC NOTES: Rapid Shell vial cell Cultures used to detect many virus in which the
sample is centrifuged onto a single layer of cells and viral growth is measured by antigen
detection methods.
PROCESSING BASED ON SPECIMEN TYPE
Laboratory maintain a menu of individual virus and algorithm for their use rather than using one
test battery for all situations.
To optimize viral detection, an algorithm for the process should be based on the type of the
specimen or the specific virus suspected.
To enhance the recovery and detection one must consider this following specimen:
o Lip and Genital Specimen
Testing for HSV, VZV, enterovirus
Urine
o Testing for CMV using Shell vial culture or Molecular assay
Stool
o Rotavirus Children <5.yo fall, winter and spring
o Enteric Adenovirus Children <5 y.o all seasons
o Enterovirus (aseptic meningitis) all ages summer and fall
o Adenovirus Gastroenteritis *specific geographic area*
NOTE: Routine testing are necessary for endemic areas.
Stool for enterovirus should be collected in conjunction to throat and CSF
specimens
Respiratory Tract
o The specimen should be separated based on patients age and medical conditions
o Immunocompromised patient requires comprehensive detection assay
o Immunocompromised patient should be examined for influenza virus during
November to April using PCR or culture.
o Children less 10 y.o requires full respiratory virus panel. (RSV, HSV, Parainfluenza,
influenza and adenovirus since they are prone to serious infection.
o Infants less 2 y.o are vulnerable to RSV Bronchitis must require hospitalization
and comprehensive supporting care. A rapid nonculture RSV assay such as PCR,
also FA staining are appropriate in these situations.

SPECIAL: Specimen for NEONATAL Patients

o Infant specimen should be examined comprehensive detection assay.
o Molecular test will determine if viral agent (e.g. Enterovirus) may be a perinatal or
congenital disease.
CSF
o In molecular assay- HSV, HIV and enterovirus
o Antibody Testing - Arbovirus
o Other can be detected infrequently including CMV, VZV, JCV and many more.
BLOOD
o CMV, VZV, HCV, adenovirus, enterovirus
PROCESSING BASED ON REQUEST FOR SPECIFIC VIRUS
ARBOVIRUS
o Diagnosis of arbovirus encephalitis, such as Eastern, Western, Venezuelan, St. Louis,
and California encephalitis, and also La Crosse and West Nile virus infection, requires
detection of virus-specific IgM antibody in serum or a rise in IgG antibody titer in
paired sera. Detection of virus-specific IgM in CSF is available for most agents.
o NOTE: Culturing Arbovirus is not practical
o NOTE: Serodiagnosis is more sensitive than PCR since Arbovirus and its nucleic acid
are detectable only during the course of infection for a brief period

CYTOMEGALOVIRUS
o CMV can be detected in almost all assay
o CMV produces Cytopathic Effects (CPE) in diploid fibroblast cells in 3-28 days. (7days)
 o CMV shell vial assay sensitivity is equivalent to conventional cell culture, and results are
available within 16 hours.
o DIAGNOSTIC NOTE: Antigenemia immunoassay uses monoclonal antibody in an
indirect immunoperoxidase or indirect immunouorescent stain to detect CMV protein
(pp65) in peripheral blood leukocytes.
o The antigenemia assay requires 3 to 5 hours and includes the sedimentation and
separation of leukocytes, counting of leukocytes, and a standardized density smear
preparation, followed by staining and counting of infected (uorescing) cells.
o Results reported as the number of positive leukocytes per total Leukocytes.
o NOTE Molecular assay have replaced Antigemia Immunoassay in some laboratories.

ENTEROVIRUS
o Enterovirus grows in Primary Kidney cells but some grows faster in Diploid Fibroblast,
buffalo green monkey kidney, or rhabdomyosarcoma cell lines.
o To reduce waste and provide availability when needed, frozen ready cells may be
used. Ready cells may be stored for up to 5 months.
o DIAGNOSTIC NOTE: CPE may use for presumptive diagnosis.
o DIAGNOSTIC NOTE: FA Stain for definitive diagnosis.
o DIAGNOSTIC NOTE: PCR preferred to use for Enterovirus septic Meningitis.
o Enterovirus commonly detected between June and December.
o Requires 4 days for detection of cell culture.

EPSTEIN-BARR VIRUS
o Serology testing is preferred for the EBV assoc. disease including Infectious
Mononucleosis.
o Cultured B-Lymphocyte used for the Isolation of EBV (not commonly performed).

HEPATITIS VIRUS
Hepatitis detected using Serologic, antigen detection or PCR.
Hepatitis A transmitted thru food and water. Detection thru IgM antibody
Hepatitis B- transmitted thru blood borne, vertical transmission
o Produces acute and chronic inflammation
o Correlated to hepatocellular carcinoma
o HBsAg (Surface antigen) a first marker for acute inflammation.
o HBsAB a marker when the infection resolves.
Hepatitis C blood borne (same as HBV)
o HCV infection becomes chronic in more than 80 % of the patients.
o HCV RNA is measured in PCR and also to assess treatment.

HERPES SIMPLEX VIRUS

o MRC-5 or Mink Lung Fibroblast recommended.
o A-549, Ready cells are also used for cultivation
o In Genital isolates; 50% of detection within 24 hrs.
100% of detection in 3-5 days
o NOTE: Isolate must be examined daily and reported as negative after 5 days.
o Real time-time PCR detects HSV within hours. Sensitive than Cell culture.
o DIAGNOSTIC NOTE: Enzyme linked viral induced system (ELVIS) Detection when
Beta-galactosidase accumulates in the cells by staining that result a blue color change
in the inverted microscope; detection within 24 hr.
o Other tests: Herpeselect, Isothermal nucleic-acid based test
o BioHelix assay using Helicase dependent amplification (HAD) can detect within 1.5 hrs.

HUMAN IMUNODEFICIENCY VIRUS and other retrovirus

o HIV 1 is detected by antigen, antibody, and reverse transcriptase PCR.
o HIV 1 and HIV 2 is also detected by Enzyme-Linked Immunosorbent Assay
o DIAGNOSTIC NOTE: Western Blot used for confirmatory testing
o RT-PCR assay is use for recently infected or not seroconverted patients.
 o Blood for transfusion is screened for antibody indicative of infection with HIV-1,
HIV-2, and human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV 2).
HTLV-1 ELISA screening tests also detect antibody to HTLV-2.
o HIV antigen (p24) test is performed to determine whether donors have been
recently infected

PEDIATRIC RESPIRATORY VIRUS

o Inuenza and parainuenza viruses, RSV, and adenoviruses are the commonly
detected from hospitalized from children 10 years old to infant.
o All viruses can be detected by uorescent staining of respiratory secretions or
rapid cell culture (shell vial).
o If fluorescent staining is negative, Cell culture will be the confirmatory test when
children suspected to virus other than RSV.
o DIAGNOSTIC NOTE: R-Mix cells in a rapid shell vial format to detect respiratory
viruses which Two R-Mix shell vial tubes are inoculated for each specimen. After
an 18- to 24-hour incubation, the cell mixture from one tube is stained with a
pooled antibody reagent designed to detect all common respiratory viruses.
Positive (uorescent) specimens have the second tube scraped, spotted onto
eight-well slides, and stained with individual antibody reagents to identify the
specific virus. R-Mix Cell is commonly used for respiratory virus .
o But for Conventional method, primary Monkey Kidney (PMK) cells by CPE or
hemadsorption used to detect parainfluenza and influenza virus.
o HEp-2 Cell culture used for Adenovirus and RSV, confirmed by FA.
o Note: Membrane ELISA are less sensitive than Cell culture but convenient for
STAT testing and result is available for 1 hour.
GASTRIC VIRUSES
o Electron Microscopy used for the detection of Viral Gastroenteritis.
o EM is intensive, Alternatively, Immunoassay for Rotaviruses and Enteric
adenovirus type 40 and 41 are commercially available.
o Norovirus and Astrovirus does not cause life-threating diarrheal disease.

TORCH (Toxoplasma, Rubella, Cytomegalovirus, Herpes Simplex Virus)

o Testing TORCH is essential for pregnant women because it can result
transplacental infection which lead to Congenital and postnatal complication.
o CMV is frequently associated to congenital infections

VARICELLA-ZOSTER VIRUS
o VZV Virus is A DNA type virus which can cause Varicella or chicken pox then
when it establishes latency in a dorsal nerve root ganglion, month to years later
varicella will reactivate and can cause Zoster (a limited form of varicella).
o Varicella (vesicular eruption) is the clinical presentation associated with a primary
VZV infection
o When vesicle use for detection, it must be a newly opened crust. (see Tzanck test)
o Cell culture can also detect VZV using Fibroblast Cell (MRC-5), requires 28
days to see a cytopathic effects (CPE) if positive.
o Shell Vial Assay can also be used with less detection time around 48 hrs. It is
used when the cell culture fails to produce CPE.
o PCR is the most sensitive for VZV but if its unavailable, FA staining is
recommended.

VIRUS
VIRUSDETECTION
DETECTIONMETHODS
METHOD
1. CYTOLOGY AND HISTOLOGY
Detection thru examination of viral inclusion and syncytia.
NOTE: in Pap-or Giemsa-stained-cytologic smears are examined for the inclusion and
syncytia.
 Hematoxylin and eosin stain can detect inclusions such as CMV, Parvovirus, Adenovirus,
Papillomavirus, and moluscum contagiosum.
Infrequently, Measles and rabies detected by examining stained smears
Rabies virus inclusion in brain tissue are called negri bodies.
BOTTOMLINE: It is less sensitive than culture but helpful to those viruses that is
dangerous and difficult to isolate such as parvovirus and rabies.

2. ELECTRON MICROSCOPY
Helpful for detecting virus that does not grow rapidly in cell culture and works best at least
106 to 107 particles per milliliter but it is very labor intensive and relatively insensitive.
Immune EM allows for visualization for detection of antibody-bound aggregate that uses
specific antiserum for virus especially if its too small for easy direct detection.
Gastroenteritis viruses and Encephalitis causing viruses (HSV, Measles, and JCV) are
far more detectable by EM in infected tissue.
Useful for detection of new viruses.

3. IMMUNODIAGNOSIS (ANTIGEN DETECTION)

Includes Fluorescent antibody, enzyme immunoassay, latex agglutination, and
immunoperoxidase test which detect viral antigen in patient specimen.
A. Direct Immunofluorescent (DI) testing involves the use of a labeled antiviral
antibody; the label is usually uorescein isothiocyanate (FITC), which is layered over a
specimen suspected of containing a homologous virus
BOTTOMLINE: More rapid and specific but less sensitive.
Suitable for large quantities of virus suspected.
B. Indirect Immunofluorescent (II) is two-step test in which unlabeled antiviral
antibody is added to the slide, followed by a labeled (FITC) antiglobulin that binds to the
first-step antibody bound to virus in the specimen.
BOTTOMLINE: Sensitive than Direct but less Specific, it takes time.
Suitable for Less quantities of virus suspected
NOTE: The increased sensitivity of indirect immunouorescence results from
signal amplification that occurs with the addition of the second antibody.
NOTE: Signal amplification decreases specificity by increasing nonspecific
background uorescence.
o False positive results (DI, II) when specimen containing yeasts, certain bacteria,
mucus, leukocytes, and other that contains FC receptor for antibody.
C. Immunofluorescent Stain
o Use for the detection of RSV, inuenza and parainuenza viruses, adenovirus, HSV,
VZV, and CMV.
o A pool of antibodies can be used to screen a specimen for multiple viruses.
o A positive screen is tested with each individual reagent to identify the exact virus.
o Successful detection when used for respiratory viruses in children but not in the
case of adult since of the lower virus particles present on them.
D. Enzyme Immunoassay
.
I. Solid-Phase ELISA
Solid-phase ELISA is performed in a small test tube or microtiter tray.
Breakaway strips of microtiter wells are available for low-volume test
runs.
II. Membrane-Bound Enzyme-Linked Immunosorbent Assay
Develop for low-volume testing and rapid result is needed (it takes 30
mins to complete).
The membrane method uses a handheld reaction chamber with a cellulose-
like membrane. Specimen and reagents are applied to the membrane
Color reaction occurs on the surface of the membrane and is read visually.

o Rotavirus- Solid-Phase ELISA

 o RSV - Both
o Influenza - Membrane-Bound Enzyme-Linked Immunosorbent Assay

BOTTOMLINE: Advantages of enzyme immunoassay is stable and can be

interpreted quantitatively (titer result) and qualitatively (degree of color). Interference
can be a problem,
o Yes it is sensitive but the quality of the result cannot be evaluated just like the
number of cells cannot be assessed unlike for FI stain that can be evaluated
microscopically. Trust the machine nalang.
E. Immunoperoxidase staining
Immunoperoxidase staining is commonly used to stain histologic sections for virus
but is less popular than immunouorescence staining in clinical virology laboratories.
F. Latex agglutination
Easy and inexpensive method but lacks sensitivity compared with ELISA and
uorescent immunoassays. (see your turgeon IS \_()_/)

4. ENZYME-LINKED VIRUS-INDUCIBLE SYSTEM

(ELVIS) uses a baby hamster kidney (BHK) cell culture system with a cloned (added)
beta-galactosidase gene that is expressed only when cells are infected with a virus.
After inoculation and overnight incubation, growth of HSV results in production of the -
galactosidase. -galactosidase serves as the reporter molecule.

5. MOLECULAR DETECTION
A. Nucleic Acid Probes
Nucleic acid detection uses nucleic acid probes, which are short segments of DNA
that hybridize with complementary viral DNA or RNA segments.
The probe is labeled with a uorescent or chromogenic tag that allows detection if
hybridization occurs.
NOTE: The probe reaction can occur in situ, such as in a tissue thin section; in liquid; or
on a reaction vessel surface or membrane.
B. Polymerase Chain Reaction
PCR is useful if the specimen is too few in number because DNA of target fragments of
Virus can be detected by probes (detection) and being amplified (amplification) by
duplicating the short DNA targets by a million-fold.
As the product is being accumulated, fluorogenic probes or fluorescent dyes used to
monitor and quantitated by the special thermal cycles with precision optics that can
monitor the fluorescence emission of the sample.

Real-time PCR -detection of DNA and the amplification occur simultaneously

Conventional PCR- detection of DNA and the amplification occur separately.

Note: Reverse Transcriptase PCR (RT-PCR) if RNA virus aims to be detected.

o The first step in RT-PCR includes making a complementary DNA strand of the
RNA segment in question.
o The usual PCR steps used to multiply the DNA target are then performed,
leading to DNA amplicons that, when identified, signify the presence of the
original RNA sequence.

6. CELL CULTURE
 A laboratory test in which samples are placed with a cell type that the virus being tested
for is able to infect. If the cells induce CPE, then the culture is Positive.
To detect virus from cell culture, one must consider the suitable host cell, cell culture
media and the technique in cell culture maintenance.
Cell culture or tissue culture starts from few cells then grow into a monolayer.
The cell must be keep moisty, supplied with nutrients and immense in culture medium.
Cell culture routinely incubated in a roller drum that rotates 0.5 to 1 rpm tilted 5 -7
degrees at 35-37 degree Celsius.
It may be incubated at stationary rack rather than roller drum.
The culture medium must maintain 7.2 pH (Physiologic levels) by using Sodium
bicarbonate as a buffer since virus produce CO2.
Phenol Red is use as an indicator of Ph |RED-normal|Yellow-Acidic|Purple-Alkaline|
Specimen is inoculated for 1-4 weeks (depending on the virus) but must be inspected
periodically by the inverted light microscopedetection for positive result by the
presence of dead cells known as Cytopathic effects (CPE).
a. 1+ 25 %
b. 2+ 50%
c. 3+ 75%
d. 4+ 100%
o Cytopathic effects presents two other important consideration aside from the (1.)
degree of CPE stated above (grading)
2. Rate at which CPE progress
E.g. HSV Cytopathic effect progresses rapidly but other two HSV
subtype, VV, and CMV grow slowly in HDF cells.
3. Cell culture in which the virus grows
e.g. Poliovirus and echovirus produce similar CPE in Primary rhesus
monkey (RMK) but echovirus does not induce CPE in continuous cell
line.
One must know the CPE is induced by the Virus itself or it is just only a natural cell death
due to old cell used, Bacterial/fungal contamination and toxic agents.
Inoculation into fresh cells should amplify viral effects and dilute toxic effects.

Composition of Cell Culture

There are two kinds of media, growth medium and maintenance medium, are used for
cell culture.
Both are prepared with Eagles minimum essential medium (EMEM) In Hanks or Earles
balanced salt solution (HBSS or EBSS, respectively) and include antimicrobials
(vancomycin 10 g/ml, amphotericin 2.5 g/ml, and gentamicin 20 g/ml) to prevent
bacterial contamination and serum protein.
o HBSS has better CO2 buffering capacity, whereas EBSS has better buffering
capacity in ambient air.
Growth medium- aside from the previously composition used both found in 2 kinds of
media, the difference of the growth medium contains more serum (10%) than the latter.
o Newborn Agammaglobulinemic calf serum is used as the serum protein because
it is contains no antibody inhibitor and free of mycoplasma than the old ones.
o It is used to provide growth of cells or to remove old cell feeding tube when the cell
culture have Incomplete monolayer.
Maintenance medium- From what stated above it contains less Serum protein (0-2%) than
the growth medium in order to keep the cell in a steady state of metabolism.
Kinds of Cell Cultures
1. Primary Cell line- Primary cell lines have been passed only once or twice since
harvesting further passage of primary cells results in a decrease Receptivity to viral
infection. (e.g., PMK cells).
2. Diploid Cell line- Diploid cell lines remain virus sensitive through 20 to 50 passages.
(e.g., HDF cells, such as lung fibroblasts)
3. Continuous Cell line- can be passed and remain sensitive to virus infections
indefinitely. Unfortunately, most viruses do not grow well in continuous cell lines.
 (E.g. human epidermoid carcinoma (HEp-2))
NOTE: A combination frequently used by clinical laboratories is RMK cells, MRC-5 lung
fibroblast cells, and HEp-2 cells or A-549 cells

Inoculation of Cell Cultures

o Inoculated cell cultures should be incubated immediately at 35C.
o After allowing virus to adsorb to the cell monolayer for 12 to 24 hours, the remaining
inoculum and culture medium are removed and replaced with fresh maintenance
medium cell to prevent culture toxicity.
o Incubation should be continued for 5 to 28 days, depending on the suspected agent.
o Maintenance medium should be changed periodically (once or twice/day) to
provide fresh nutrients to the cells.
o When there is no CPE occur or ambiguous CPE (toxic/bacterial/fungal induced), the
cell and fluid from the first tube will be transferred to another (2 nd) tube. That process
known as Blind passage. It is performed to dilute the toxic coming from the first
tube. It is rarely performed nowadays.
7. SHELL VIAL CELL CULTURE
A rapid modification of conventional cell culture because the infected cell monolayer is
stained for viral antigens (using virus-specific immunouorescent conjugates) produced
soon after infection (1-2 days), before the development of CPE.
It place in a shell vial culture tube, a 15 45 mm 1-dram vial, is prepared by adding a
round coverslip to the bottom of the tube, covering this with growth medium, and adding
appropriate cells.
During incubation (placed on the low-speed roller rack), a cell monolayer forms on top
of the coverslip. Shell vials should be used 5 to 9 days after cells have been inoculated.
uorescing inclusions are used to confirm the presence of an infecting virus.
It is best used for viruses requires long incubation time to form CPE such as CMV and
VZV because most other viruses are detected within 24 hrs.
NOTE: The disadvantage is that only a single type of virus can be detected per shell vial.
Other strategies pool antibody for detection of many viruses with a single vial.
8. OTHER TEST USING CELL CULTURE
A. CPE Description
Cytopathic effects is distinct from every different viruses, just as the colonies of bacteria in
agar plate which leads to presumptive identification.
Virus-infected cells change their usual morphology and eventually lyse or detach from the
glass surface while dying
Parameters for preliminary (presumptive) identification of Virus thru CPE.
Cell line used
how quickly the virus produced CPE
description of the CPE (table below)
Fluorescent-labeled antisera (FA), available for most viruses, are used for confirmation.
NOTE: For the case of to differentiate Rhinovirus from Enterovirus, Acid Lability is used if
theres no Fluorescent-labeled antisera available.
Virus CPE Description Identification and Comments
Adenovirus Rounding and aggregation of infected cells in grapelike clusters Confirm by FA test; serotype by cell culture neutralization

Cytomegalovirus Discrete, small foci of rounded cells Distinct CPE sufficient to identify; confirm by FA test
(CMV)
Enterovirus Characteristic retractile angular or tear-shaped Confirm by FA test; stable at pH 3
CPE; progresses to involve entire monolayer
Herpes simplex Rounded, swollen refractive cells; occasional syncytia, especially Distinct CPE sufficient to identify; confirm by FA test
(HSV) with HSV-2; rapidly involves entire monolayer

Inuenza Destructive degeneration with Detect by hemadsorption or hemagglutination with guinea

swollen, vacuolated cells pig RBCs; identify by FA test
Mumps CPE usually absent; syncytia Detect by hemadsorption with guinea pig RBCs; confirm by
occasionally seen FA test
Parainuenza CPE usually minimal or Detect by hemadsorption with guinea pig RBCs; identify by
 absent FA test

Respiratory Syncytia in HEp-2 cells Distinct CPE in HEp-2 cells sufficient for presumptive
syncytial virus identification; confirm by FA test
(RSV)
Rhinovirus Characteristic refractile rounding of cells; in PMK, CPE is identical Labile at pH 3; growth
to that optimal at 32 to 33C
produced by enteroviruses
Varicella-zoster Discrete foci of rounded, swollen, refractile cells; slowly involves Confirm by FA test
virus entire monolayer

B. Hemadsorption Test
It is where positive if the RBC adsorb to the infected cell membrane due to its affinity to
hemagglutinins which is present in the host cell.
Guinea pig red blood cells (RBCs) is added to the cell culture tube, followed by a
wash to remove no adsorbed RBCs, results in a ring of RBCs around infected cells
Cell cultures demonstrating hemadsorption can be stained with uorescent-labeled
antisera to identify the specific hemadsorbing virus present (confirmation).
It is greatly used if the virus produce little or no CPE (e.g., inuenza, parainuenza, and
mumps viruses)

9. SEROLOGIC TEST

It was the primary means of laboratory diagnosis of viral infections until the mid-1970s
Today, the primary purpose of serologic test is to determine immune status and to confirm
the diagnosis of infection when the virus cannot be cultivated in cell culture or detected
readily by immunoassay or molecular assays.
In most viral infection, IgM will first to rise on acute infection then IgG will takes place and
IgM will be undetectable after 1-4 month after the infection resolves, but if the virus
reactivate from latent stage, IgG will rise 4x (increase titer result) and IgM will now
difficult to detect. This body response will make IgM and IgG as a marker for active and
recent infections.
o Virus-specific IgM detection in an acute-phase specimen collected at least 7 to 14
days after the onset of infection indicates current or very recent disease.
Detection of a fourfold antibody titer rise between acute and convalescent
sera (collected 2-3 weeks after acute-phase specimen) also indicates current
or recent disease.
o Virus specific IgG- is used when the patient is recently infected that kind of virus or
theres a result of the convalescent specimen.
IgG tests are not needed on the first, acute specimen until receipt of the
convalescent specimen. This eliminates useless testing of single specimens
when a second sample is never submitted for analysis.
TECHNIQUES USED FOR SEROLOGICAL TESTS
Complement Fixation (CF)
o Laboratory intensive, technically demanding method best ftted to batch testing.
Enzyme linked- Indirect Immunouorescence (ELISA)
o Can be used to detect IgM-specific antibodies free of common interfering factors
o It is separated by using antibody-capture technique.
Indirect Immunofluorescence
o best used for individual specimens or small-batch testing
o But it requires prior separation and elimination of the IgM from IgG fraction.
o It is separated by using on exchange chromatography, by immune precipitation, or
with an IgG inactivation reagent such as Gullsorb which contains caprine (Anti-IgG).
o Still Indirect immunofluorescence is prone to False (+) due to IgG.
Anticomplement Immunouorescence (ACIF)
o It is an alternative technique since Indirect Immunofluorescence is prone to false (+)
results.
o Because uorescent-labeled complement binds only to antigen-antibody complexes,
the nonspecific antibody attached by Fc receptors, which is complement free, does
not uoresce.
 Western Immunoblotting
o Complex antigens are separated into individual components during the Western blot
procedure, and positive or negative reactions are observed.
o Western blot provides more specific result than other serologic tests, such as EIA.

NOTE: Rheumatoid factor can cause false (+) of result

Strongly Binding IgG that prevent binding IgM cause false (-) result.

IMMUNE STATUS TEST

Used to detect patients who have been infected with (or vaccinated for) a virus in the
past, conferring lifelong immunity to reinfection.
Examples
o Rubella antibody immune status testing is used with women of childbearing age.
A positive result of the presence of IgG antibody indicates past infection or
immunization and implies that congenital infection will not occur during
subsequent pregnancies.
Absence of IgG antibody implies susceptibility to infection and should prompt
rubella vaccination if the woman is not pregnant.
o Varicella and Measles; if the person has low IgG for that virus means they must
avoid patient who suffer that kind of condition.
o Cytomegalovirus immune status testing is needed when performing organ
transplant or blood transfusion and it is essential for newborn screening.

SEROLOGY PANELS
Clinical Virology Laboratories must have a method for

Storing (can be done by Freezing in liquid nitrogen at 70C)

Retrieving Viruses ( for control strains and epidemiologic investigation)
Accurate inventory system

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l.mendiola