MT MYCOLOGY & VIROLOGY

6322 LABORATORY SHIFT 3

GENERA
UNIT 7: LABORATORY DIAGNOSIS FOR
Orthobunyavirus LCM
NEGATIVE-SENSE (-) ssRNA VIRUSES Phlebovirus Agents of Lassa Fever
Nairovirus
Sources:
Hantavirus
Dr. Miguel Francisco C. Cajipe, MD, RMT, DPSP-CP and Mr. Jerry
Arboviruses Robovirus
F. Ching, Jr., RMT, MSMT’s Powerpoint presentation and Lecture
(Arthropod-borne) (Rodent-borne)
Discussion
NOTES
● SEGMENTED GENOME: enables the generation of
OUTLINE
reassortant viruses
PRE-TEST 1 ○ Mixing or reshuffling of nucleic acids during the
NEGATIVE-SENSE RNA VIRUSES 2
ARENAVIRIDAE 2 replication or morphogenesis segment → new
BUNYAVIRIDAE 2 combination of RNA segment
RHABDOVIRIDAE 4 ○ Results in a new property in the virus → diversity
Antemortem Diagnosis 4
Postmortem Diagnosis 5
FILOVIRIDAE 6 LASSA FEVER GROUP
ORTHOMYXOVIRIDAE 8 ● Junin Virus
PARAMYXOVIRIDAE 9 ● Machupo Virus
Respiratory Syncytial Virus 9 New World
● Guanarito Virus
Parainfluenza Viruses 11
● Sabia Virus
Metapneumovirus 11
Rubeola Virus (Measles Virus) 11 ● Lujo Virus - isolated from Africa, Europe
Old World
Rubulavirus (Mumps Virus) 13 and Asia
Hendra And Nipah Virus 14
POST TEST QUESTIONS 15
ARENAVIRIDAE
_____________________________________________________
(ROBO VIRUSES)
PRE-TEST QUESTIONS OVERVIEW
1. (True/False). Arenaviridae are recommended to be grown in ● Greek word Arenanosa means “sandy”
culture. ○ Pebbly, sandy and grainy or granular appearance
2. The CPE of Rhabdoviruses are best visualized using ○ Due to the presence of ribosomes
Hematoxylin and Eosin stain. ● Causative agent for hemorrhagic fever syndrome
3. The best samples for the diagnosis of Influenza virus are from ● Segmented, helical, enveloped
the nasopharynx? ● T-shaped or club-shaped spikes
4. Warthin-Finkeldey cells are characteristic of Rubeola or ● Infect rodents and humans
Measles virus.
5. Give one specimen where the Mumps virus can be found: MODE OF TRANSMISSION
Saliva ● Inhalation of aerosols and rodent excrements
● Direct contact with infected rodents
NEGATIVE-SENSE RNA VIRUSES ● Contact with fomites
“FORBAP”
Filovirus Bunyavirus LABORATORY DIAGNOSIS
Orthomyxovirus Arenavirus
● NOT RECOMMENDED: Cell Culture
Rhabdovirus Paramyxovirus
● Lymphocytic Choriomeningitis (LCM) and Lassa virus
○ Dangerous to grow in a cell culture
COMPARISON OF SINGLES STRANDED (-) SENSE RNA
○ Highly contagious virus
ARBOVIRUSES & ROBOVIRUSES
○ BSL-3 for LCM virus
BUNYAVIRIDAE ARENAVIRIDAE ■ Required biosafety level to culture LCM virus
(-) ssRNA SEGMENTED GENOME ○ BSL-4 for Lassa Virus
Triple segmented (L, M, S) Duo segmented (L, S) ■ Required biosafety level to culture Lassa virus
Both are ZOONOSES
Causative agents of CNS diseases or hemorrhagic fever

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

● SPECIMENS DETECTION OF ANTIBODIES

○ Throat specimens ● Documenting acute infection
○ Urine samples ● Recent infection usually 5 days from the
IgM
■ Used for Lassa Fever Virus but NOT for LCM onset of illness might detect the IgM
○ Must be processed in either a BSL-3 or BSL-4 depending antibody
on the expected finding ● If more than a week, you can detect IgG
■ Laboratory should be ideally forewarned when ● Seroconversion of four (4) fold rise in
processing specimens IgG
● DIAGNOSIS ○ If you see a four (4) fold rise in IgG,
○ Dr. Cajipe: “I’ve read the books and so far, I have not IgG that is an indicative of recent
found a book explicitly stating that there is a test infection
registered for LCM virus to detect IgM and IgG ● Observed in paired samples
antibodies. However, I’m assuming that there is also a ○ 1 sample taken during acute
test that will detect IgM and IgG antibodies against LCM ○ 1 sample taken during convalescent
virus” OTHER SEROLOGIC METHODS
POLYMERASE CHAIN REACTION (RT-PCR) Viral
Neutralization For identification of the virus
● Most sensitive test for detecting Arenaviruses Assays
SEROLOGY ● In case of intense viremia associated
with the following:
● More rapid diagnosis
○ Rift Valley Fever
● IgM and IgG antibody detection for Lassa virus
ELISA Antigen ○ Hanta Infection
○ Detection of antigen or antibody
Test ■ Like hemorrhagic fever with
renal syndrome
BUNYAVIRIDAE
○ Crimean-Congo Hemorrhagic
(ARBOVIRUSES)
Fever
OVERVIEW
● Arthropod borne viruses [Sir Alvin]:
● Basically transmitted by insects like mosquitoes and tick
● For DENGUE, we can detect antigens in the blood
● MANIFESTATIONS or SYNDROMES:
○ Antigens can serve as an early marker
○ Fever with muscle pain or myalgia
■ NS1: early marker for dengue
○ Fever with or without hemorrhagic rash
■ This can already be (+) positive even before the
○ Encephalitis (EEE, VEE, WEE)
detection of IgM
○ Hemorrhagic fever
○ Very useful in the first few days of the illness
● Can affect humans and animals
■ Usually 9 or 10 days of the illness
○ Horses and birds can also be affected
○ In the early days of infection, if the patient is negative,
the NS1 antigen testing can be used. If fever persists
LABORATORY DIAGNOSIS for 3 days, the NS1 antigen can result to positive.
POLYMERASE CHAIN REACTION (RT-PCR) ○ Sometimes the platelet count is not reliable,
sometimes the platelet is okay and positive ka na pala
● Most sensitive test, detecting the genome for this virus sa NS1
● High specific and sensitive test to identify viruses

SEROLOGIC METHOD PLAQUE REDUCTION NEUTRALIZATION TEST (PRNT)

● Detection of antibodies ● Used to confirm the serologic test

● Most common method to diagnose arboviruses
● Detection of IgG or IgM antibodies which depends on the
arbovirus
● (Sir Alvin) More rapid but there is a question if it is as
sensitive as PCR
● After the presence of neutralizing antibodies
● (+) Positive Result:
○ Plates with plaques of the virus and then you would add
the serum sample and determine the titer
■ Titer: highest dilution that shows 70% reduction of
the plaques

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

OTHER METHODS
● NAAT (PCR) RIFT VALLEY FEVER VIRUS
● CULTURE: not usually performed (Genus Phlebovirus)
● ANTIGEN DETECTION
● VIRAL ISOLATION AND EXAMINATION: using electron [LONG ARROW]:
microscope Exhibiting subunits with
a central hole
CONSIDERATIONS IN PRESUMPTIVE DIAGNOSIS
(Particularly in febrile illnesses associated with the infections
caused by the virus)
● Geographic site [SHORT ARROW]:
● Season Regularly spaced
● Kind of arthropod pressen in the area subunits

Bar, 100 nm
BUNYAVIRIDAE VIRUS

Virions have
moderately dense
centers and accumulate
in the cisternae of the
Golgi complex of a Rift
Valley fever
virus-infected cell.

Bar, 500 nm
CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS
(Genus Nairovirus)
ORTHOBUNYAVIRUS
(La Crosse Virus) [LONG ARROW]:
RT-PCR Target M-segment polyprotein genes Small surface subunits
Serology Serum or CSF IgM
Antigen Glycoprotein-1
[SHORT ARROW]:
Appear as a peripheral
ELECTRON MICROGRAPH
fringe
OF THE DIFFERENT MEMBERS OF BUNYAVIRIDAE
Bar, 100 nm
HANTAAN VIRUS
ANHERI VIRUS
(Genus Hantavirus)
(Genus Orthobunyavirus)

Knob-like surface
Grid-like surface structures
pattern

Bar, 100 nm

Bar, 100 mm

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

SIN NOMBRE VIRUS PARTICLES CLINICAL INVESTIGATION

(Genus Hantavirus) ● It uses computed tomography (CT) and magnetic image
resonance (MRI)
○ MRI images of the brain of the infected host would show
● Extracellular that the part of the brain that is usually infected by the
● Have a variety of virus is the brain stem region and the hippocampus
sizes and internal ○ Expensive
cores composed of
thin thread-like LABORATORY INVESTIGATION
material ● EEG (electroencelophatograpy)
○ Detects brain waves
Bar, 500 nm
○ Shows non specific abnormalities

HANTAVIRUSES ANTIGEN DETECTION ASSAY

LABORATORY DIAGNOSIS DIRECT
● No approved laboratory testing as of this writing FLUORESCENT Highly sensitive and specific
○ In spite of this RT-PCR tests are being done AB TEST
● Serologic tests CORNEAL ● Can be examined by
IMPRESSION treating/subjecting the sample
HANTAVIRUS
SMEAR - BY FAT through fluorescent antigen test
RT-PCR TARGET N Gene
● Similar to touch smear
SEROLOGY ELISA (IgG and IgM)
ANTIGEN Nucleocapsid Protein PROCEDURE
1. Touch the infected cornea with a
RHABDOVIRIDAE sterile slide, carefully to avoid
OVERVIEW causing abrasion to the eye of the
● Bullet-shaped appearance host.
● Usually done post-mortem 2. Smear produced is stained with a
○ Central Nervous System fluorescent dye
■ Prefer the brain itself 3. Appearance of round to oval
○ Detection of negri bodies in neurons intracytoplasmic inclusions in
■ Usually found in the Hippocampus or Hippocampal corneal epithelial cells
area, that is somewhat near the middle or brain HAIR FOLLICLE ● Nape is where you can isolate a
stem. OF NAPE OF substantial amount of the virus
■ Although they can be found in the other areas of the NECK ● Acquire 10 hair follicles for
brain, studies just show that these neurons with specimen
negri bodies are present in the hippocampal area ○ Get the cutaneous nerve
which makes the pathologist's life easier since they ● Site of infection of rhabdovirus
will concentrate because they are there a lot ● For Rabies viruses
VIRAL CULTURE ● Use vero cells
ANTEMORTEM DIAGNOSIS ○ For Marburg strains or species
(Ma’am Domingo) ANIMAL ● By using guinea pig for Ebola
● Because it shows slow waves not characteristic of brain INOCULATION Virus
waves, such as alpha-, delta-, and gamma-waves ● Greater chances to recover an
● Biological Specimens: Usually sterile Ebola virus
○ CSF ANTIBODY TEST ON CSF
■ Prioritize CSF specimens when processing samples MOUSE ● Requires an intracerebral
○ Tissue NEUTRALIZATION inoculation of mouse
■ E.g., skin TEST (MNT) ● Inoculate the virus intracerebrally
○ Serum into the cerebrum
○ Saliva ● Found to be very useful for Rabies
● Viral Isolation: animal inoculation (e.g. mice) virus diagnosis

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

● Superseded enzyme immunoassay

technology NEGRI BODIES
● Recommended by WHO as a
standard test
● If MNT test is not possible, a
suitable substitute would be the
RFFIT test
RAPID ● Also follows a neutralization reaction
FLUORESCENT ● Found to be very useful for Rabies
FOCUS virus diagnosis
INHIBITION TEST ● Superseded enzyme immunoassay
(RFFIT) technology
FLUORESCENT ANTIBODY VIRUS NEUTRALIZATION TEST
(FAVNT)
INDIRECT FLUORESCENT ANTIBODY TEST (IFA)
● Remember it is cytoplasmic
[NOTES]
○ Found in the cytoplasm, NOT in the nucleus
● CSF antibodies indicate encephalitis stage, irrespective of
● You can see the cell body, nucleus with prominent
vaccination history
nucleoli, and dendrites
● Serum antibodies appear late and are present also after
● These cells are largely found in the hippocampal area of
vaccination.
the brain but they can be found in the entire stretch of the
● For neutralization tests, specimens, especially CSF, must
CNS such as spinal cord, other parts of the brain
be diluted prior to viral suspension or culture of infected
● H & E stain is used
cells.
● Usually done in post-mortem
● Dr. Cajipe:
TISSUE CULTURE
○ The same with animals for example a dog by cutting
● BHK21 or BHKF21 (Baby Hamster Kidney Fibroblast) cell the head, then putting it in an ice bag then we will send
line it to the RITM (Research Institute for Tropical
○ Tissue culture cell Medicine) then they will look for the negri bodies
○ Cultured from a 1-day old hamster ○ Pag nakita niyo na to, pumusta na kayo na you are
● PROCEDURE dealing with Rabies virus
○ Infect the cell → Add diluted serum sample →
Neutralization reaction →
FISH-EYED NUCLEOLUS
○ How many of the viral particles were prevented from
infecting the cell line?

RNA DETECTION BY RT-PCR

● Most specific and sensitive method
● SPECIMENS:
○ Saliva
○ CSF
○ Skin

POSTMORTEM DIAGNOSIS

DETECTION OF NEGRI BODIES

● Histopathologic examination of infected brain tissue can be
used to directly identify Negri bodies in neuronal cytoplasm
○ Considered pathognomonic for rabies virus infection
○ NEGRI BODIES: Intracytoplasmic inclusion bodies that
consist of aggregates of viral nucleocapsids
[POINTED IMAGE]: Fish-eyed nucleolus of the neurons
● You see some round, oval-shaped, sharply delineated
eosinophilic inclusion
● Location:
○ Perikarya or cell body or soma of the nerve neuron
○ Proximal dendrites

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

MOUSE INOCULATION EBOLA VIRION

● Swiss Albino Mouse
○ Most susceptible, mabilis ma-infect

VIRAL ISOLATION
● From animal (MIT) cell culture
○ BHKF21
■ Rabies Tissue - Culture Infection Test (RT-CIT)

DIRECT FLUORESCENCE ANTIBODY TEST

● Direct Fluorescent Antibody Test (DFA) on brain smear
● “Gold standard test” in United States for postmortem
diagnosis

SEROLOGIC TESTING
● Serologic testing is usually geared towards monitoring
vaccination status of an individual
○ ELISA
■ Done to know if the person still needs to be
vaccinated and not used to diagnose since usually
antibodies of rabies (example) are not seen until it is
too late.
■ To monitor vaccinations only
○ Antibodies are not usually detected until late in the
disease

FILOVIRIDAE
OVERVIEW
● Considered as a pantropic virus
○ Affecting many types of body tissues and the organs
themselves
○ They have capacity to produce lesions in a number or
even every organ
○ The most affected: liver and spleen
● Just like Lassa fever, the Filoviruses (Ebola Virus) is very
dangerous to handle, the lab must be equipped with BSL3
or BSL4 in order to perform diagnostic study
● Extreme care not to have accidental infections

DIFFERENT SPECIES OF FILOVIRUS

● Ebola Virus ● Sudan Virus
● Marburg Virus ● Reston Strain
● Zaire Virus (Philippines)

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

● Ebola Virus contains: Animal inoculation ● By using guinea pig for Ebola
○ Linear Virus
○ Non-segmented RNA genome ● Greater chances to recover an
○ Single strand Ebola virus
○ RNA genomes of negative polarity Electron Microscopy
● Like all filoviruses, Ebola virus are filamentous particles
that may appear in the shaped of a “Shepherd's crook” or ORTHOMYXOVIRIDAE
in the shape of a “U” or a “6” INFLUENZA (A, B, and C)
● May be:
○ Coiled CHARACTERISTICS
○ Toroid ● Proteins present:
○ Branched ○ Glycoprotein spikes
● Median particle length ranges from 974 nm to 1086nm and ○ Hemagglutinin spike
80nm in width ○ Neuraminidase spike
● What makes this unique is because of its unique, negative
FILAMENTOUS MORPHOLOGY OF FILOVIRUS strand EIGHT (8) RNA nucleocapsid segments

STRUCTURE OF ORTHOMYXOVIRIDAE

Electron micrograph that shows the filamentous morphology
of the filoviruses, the background is negative staining
LABORATORY DIAGNOSIS
● Whatever the test is, it is highly recommended that specimens
tested for the presence of filoviruses are processed in a
BSL-4 laboratory facility.
○ Dr. Cajipe: These viruses are nosocomially acquired
most of the time, very contagious, there is an epidemic
potential so whatever the test, it is highly recommended
that these specimens that will be tested for the presence
of filoviruses must be processed in a BSL-4 laboratory
facility, it is a very high risk specimen
● Influenza A (H1N1) virus:
○ Subtype of influenza A virus
○ Most common cause of human influenza (flu) in 2009
● The different strains causing influenza or flu syndrome are
named based on the 2 glycoproteins.
A type of glycoside hydrolase enzyme
which help move the virus particles
NEURAMINIDASE
through the infected call and assist in
budding from the host cells
Causes red blood cells to clump together
HEMAGGLUTININ
and binds the virus to the infected cells

Viral antigen ● IgM and IgG detection

detection/ Ab
Enzyme ● Specimen: Body fluids
immunoassays, ○ Dr. Cajipe: can be blood,
Immunofluorescence CSF or effusions but ideally
its blood
RT-PCR ● Used to confirm diagnosis

Viral culture ● Use vero cells

○ For Marburg strains or
species

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

LABORATORY DIAGNOSIS ● Ma’am Domingo: Isolate the antigen particles of the virus
● Best diagnosed during the first 2 to 3 days of illness by ● Sensitivity: 73-95%, higher sensitivity for Influenza A
antigen based assays and culture ● Specificity: 95%
○ Dr. Cajipe: The 2nd day is the best time to collect the ● SPECIMEN
specimen ○ Nasopharynx
■ From the nasopharynx, oropharynx, a little bit of the ■ most common specimen
throat near the trachea ■ Still have good sensitivity
● Influenza virus infects the respiratory epithelium ○ Specimens from the lower respiratory tract will increase
● SPECIMEN the yield of this test
○ Upper respiratory tract specimens ■ Bronchi, near lung parenchyma
■ Swabs, washings and aspirates ■ Alveoli
○ Sample must be held only at 4°C ■ Alveolar suck
○ If more than 5 days, subject the sample to freezer ■ Terminal bronchioles
temperature (at least -70°C)
DIRECT FLUORESCENCE ANTIGEN
VIRAL CULTURE
● CULTURE OF CHOICE: Amniotic cavity of embryonated
eggs
● Viral Culture is the next most sensitive
● Shell Vial Culture: rapid diagnosis
● NO CYTOPATHIC EFFECT is produced, newly produced
virus can be identified using the following:
○ Hemadsorption
■ Newly produced virus in the medium
■ 3 to 5 days after inoculation
○ Hemagglutination
■ Culture fluid ● Shown are cells that are positive to the antigen, turning
■ 5 to 7 days after inoculation green, the fluorescent dye
● (+) Positive result: turns green
CULTURE MEDIA
Hybrid Mink Lung Very sensitive
IMMUNOCHROMATOGRAPHY
A549 Adenocarcinoma Human Alveolar
Basal Epithelial Cells ● Commercially available
PMK ● Primary Monkey Kidney ● Readily accessible
● Culture of choice ● Less sensitive compared to other tests
MDCK ● Madin Darby Canine Kidney Cells ○ Due to being seasonal
● Culture of choice ○ Influenza occurs during cold weather
[NOTE]:
● Recovery of viruses using these cell lines is slow MOLECULAR ANALYSIS (RT-PCR)
○ To hasten the recovery of these viral particles, shell ● Preferred diagnosis for influenza
vial culture, either Hybrid Mink Lung or A549, may be ● Most sensitive test
used in this type of method ● Specific and rapid (less than 1 day)
● Inoculated cell culture are incubated in the absence of
serum in cell culture is required OTHER METHODS (Ma’am Domingo)
○ Serum can contains non-specific viral inhibitory factors
and in the presence of trypsin, which cleaves and HEMADSORPTION Detect the presence of
activate the hemagglutinin hemagglutinin protein
HEMAGGLUTINATION ● For the detection of virus in
INHIBITION TEST secretions
DIRECT FLUORESCENCE ANTIBODY (DFA) STAIN
● Four-fold rise in titer to
● Can demonstrate virus in infected columnar epithelial cells conclude influenza infection
(Respiratory Epithelium) HEMAGGLUTINATION ● Not only for epidemiological
● The use of fluorescent tag antibody to detect the antigen INHIBITION TEST studies

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

● Type and strain of influenza CYTOPATHIC EFFECT

virus
ELISA ● Detection of viral antigen
● Highly sensitive and specific
FLUORESCENCE TECHNIQUE AND COMPLEMENT
[RED ARROW]:
FIXATION
Formation of syncytia due
to fusion protein
PARAMYXOVIRIDAE
OVERVIEW
● Largest RNA viruses with regards to the size
● Enveloped viruses
○ There are proteins present inside the envelope:
■ Fusion proteins
■ Neuraminidase proteins (NA) VIRAL CULTURE
■ Hemagglutinin proteins (HA) ● Replicates slowly in viral culture
● 10 days for CPE to appear

● A549 and Mink Lung Cells

● Speeds up the recovery of the virus
(According to Henry’s, it is still slow)
● Sir Alvin: Rapid modification of cell
Shell Vial culture
Culture ● Sir Alvin: You can already detect the
antigens using antibodies that are
labelled with fluorescence dyes
● Can be tested 24-28 hours later in IF or
RT-PCR
HELA Cell Line ● Highly sensitive for the cytopathic
RESPIRATORY SYNCYTIAL VIRUS effects
OVERVIEW ● Sir Alvin: Immortal cell lines
● Fragile and labile virus HEP2 Cell Line ● However this one recovers the virus
● Associated with pneumonia or respiratory illnesses slower than shell vial culture
especially for infants ● (+) giant cells and syncytial formation
● There is a possibility of reoccur and no complete immunity in cultures; also HELA cell line*

LABORATORY DIAGNOSIS SHELL VIAL TECHNIQUE

● SPECIMEN:
○ Nasopharyngeal swabs
○ Washing
● Freezing of clinical specimens may result in complete loss
of infectivity
● CYTOPATHIC EFFECT (CPE)
○ Formation of syncytial or syncytium
■ Target cells will fuse together due to fusion protein
■ Multinucleated giant cells

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES
● Rapid isolation; can be tested 24-28 hours later in IF or
RT-PCR
DIRECT FLUORESCENCE ANTIBODY (DFA) STAIN /
ENZYME IMMUNOASSAY (EIA)
● More sensitive than culture methods
● Faster results
● (+) Cytopathic Effect
○ IF, ElA, and Serum neutralization tests
● For DFA, you are after the the antigens
○ You only need to use one labelled antibody
○ The label is fluorescent dyes, such as FITC
○ With the nasopharyngeal swab, the antigens will be
detected
● For EIA, you are after the antibodies
○ Either IgG or IgM antibodies
COMMERCIALLY AVAILABLE TEST KITS
PRINCIPLE: Direct Fluorescence Antibody
DIRECT FLUORESCENCE ANTIBODY
● (+) result: Apple green fluorescence
● (-) result: Red color: cells NOT affected by the virus
OTHER METHODS: Molecular analysis
● RT-PCR:
○ The most sensitive among all the tests
○ Very expensive
○ Test of choice for respiratory secretions
● NAT:
○ Preferred method for adult specimens
● Since RSV (Respiratory Syncytial Virus) causes serious
disease especially in infants, RAPID DIAGNOSIS IS A MUST.
○ Life threatening, especially those infants who have small
tracheas that can be clogged by the plugs made by RSV
PARAINFLUENZA VIRUSES
LABORATORY DIAGNOSIS
DIRECT FLUORESCENCE ANTIBODY (DFA) STAIN / EIA
CULTURE (DFA NEGATIVE SPECIMENS)
● Shell vial culture methods using A549 and Mink Lung is
preferred
NUCLEIC ACID TESTS
METAPNEUMOVIRUS
OVERVIEW
● Also associated with respiratory illness
● Similar with Respiratory Syncytial Virus (RSV)
LABORATORY DIAGNOSIS
● Specimens: Nasopharyngeal aspirates or swabs
CULTURE
● Generally slow growth is exhibited
● Sir Jerry: Not useful clinically
● LLC-MK2 and Vero Cells are preferred if the results are not
needed rapidly
● Takes around 7 days
LLC-MK2 Cells ● 10-14 days (Sir Jerry)
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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

● (+) Cytopathic Effect: small round ● Caused by Rubeola Virus

plaques with occasional syncytia ● Childhood disease
Vero Cells Takes around 7 days
MODE OF TRANSMISSION AND PATHOGENESIS
Shell Vial Culture ● Faster recovery, but still relatively
slow ● MODE OF TRANSMISSION:
● High sensitivity among cell culture ○ Droplets
methods ○ Close contact with infected children
PMK ● SYMPTOMS:
(Primary Monkey ○ Maculopapular rashes
Kidney) ○ 3Cs:
■ Coryza
A549
■ Conjunctivitis
■ Cough
METAPNEUMOVIRUS
○ KOPLIK SPOTS
■ Hallmark or pathognomonic Sign
■ Usually diagnosed on clinical grounds

LABORATORY DIAGNOSIS
● SPECIMEN:
Uninfected ○ Epithelial cells from respiratory secretions (throat)
LLC-MK2 Cells ○ Conjunctival washings
○ Urinary sediment cells (urine)
● Rarely asked for a diagnostic test for measles
○ Largely clinical diagnosis
■ If rashes are present, measles can be diagnosed
○ In encephalitis, diagnosis is needed to make sure that
it is a complication caused by measles
■ Wrong drug prescription if not diagnosed properly

CULTURE
1

● Usually difficult for measles

Infected ○ Clinical grounds is already sufficient and enough
LLC-MK2 Cells ● SPECIMEN:
○ Nasopharyngeal
○ Conjunctival swabs
○ Blood samples
○ Respiratory secretions
○ Urine
Present CPE in metapneumovirus cell culture ● CELL CULTURE LINE used:
● Look for syncytia and find presence of small round plaque ○ Primary Monkey Kidney Cell (PMK)
○ Lymphoblastoid cell line
SEROLOGICAL TESTS ○ B95-A
● Direct and Indirect Fluorescent Antibody ■ (+) Cytopathic Effect: Seen between 7-10 days
○ Detection of viral antigens ■ Warthin-Finkeldey Giant Cells
➔ Spindle-shaped cells
MOLECULAR ANALYSIS ➔ Multinucleated giant cell containing acidophilic
● RT-PCR nuclear inclusions
○ Method of choice for children with respiratory illness ○ Vero Cell Line
○ Most superior in terms of sensitivity ● SHELL VIAL CULTURE TESTS:
● Gold standard ○ Rapid diagnosis (2-3 days)

WARTHIN FINKELDEY CELLS

RUBEOLA VIRUS (MEASLES VIRUS)
(Spindle-Shaped Cells)
OVERVIEW

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

■ Antibody titer (10 to 100-fold)

● Direct IF (Immunofluorescence) and IIF (Indirect
Immunofluorescence)
○ Measles virus Ag detection from NPS specimens
● Serum Neutralization Tests
○ Presence of neutralizing antibodies against measles

MOLECULAR ANALYSIS
● RT-PCR: detection of viral RNA; most sensitive method
● NAT (Nucleic Acid Testing): used if IgM testing is
compromised due to vaccination

RUBULAVIRUS (MUMPS VIRUS)

MODE OF TRANSMISSION AND PATHOGENESIS
● Childhood disease transmitted via droplets from the saliva
● Viral disease that leads to the inflammation of the salivary
gland such as the parotid gland
● Certain complications where other organs or tissues can be
inflamed, such as the ovaries, testes, pancreas and brain
● ORCHITIS
○ Complication among males include the inflammation of
the testes
○ May lead to sterility
■ Not in all cases
Huge syncytium that contains hundreds of nuclei
LABORATORY DIAGNOSIS

SPECIMEN
● Saliva ● Pharynx
○ Stensen’s Duct ● Urine
→ duct of parotid ● Cerebrospinal
gland ● Serum
[NOTE]:
● If the symptoms would appear less than three (3) or 5 days
after the onset, you can collect saliva
● But if more than three (3) days, you can add serum
samples
○ You are after to look at the antibody (IgM antibody)
● If there is a complication in testes, you can add urine
● H & E stain samples (2 weeks)
● Can also be seen post-mortem
● CPE of Measles virus CULTURE
● Formation of syncytia
● SPECIMENS OF CHOICE:
○ Saliva, Swabbing of Stensen’s Duct, CSF, Urine
SEROLOGICAL TESTS
○ Collected few days before the onset of illness and;
● ELISA, Hemagglutination-Inhibition, and Neutralization ○ Until 8 days after parotitis appears
tests ● Mumps grow well in primary monkey kidney media
○ Fastest among all tests ● Monkey Kidney Cell Line
○ Used for the detection of antibody titer ● Vero cell
■ Rises by four-fold between acute and the ● LLC-MK2
convalescent phase ● Shell Vial
○ Used for the detection of measles-specific IM ○ (+) CYTOPATHIC EFFECT (CPE): Multinucleated giant
○ (+) SSPE or Subacute Sclerosing Panencephalitis cells and syncytial cells

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES
○ However, NOT ALL primary mumps viral isolates show
characteristic syncytial cytopathic effects.
■ Thus, best way to diagnose or to test mumps virus
is through RT-PCR testing
● Thermolabile
○ Sample must be inoculated shortly after collection
● Simple detection of mumps antibody is not adequate to
diagnose an infection
○ Evidence of mumps infection: An antibody rise can be
demonstrated using paired sera, such as a 4-fold or a
greater rise in antibody titer
HEMADSORPTION / HEMAGGLUTINATION INHIBITION
● Most common method for detecting antibodies
● SPECIMEN: Serum sample
● Performed if no cytopathic effects are seen in the culture.
● Antigens will adhere to the RBCs
● (+) Positive Result: NO AGGLUTINATION
● Can also detect neutralizing antibodies against mumps virus
[Overview of Hemadsorption by Sir Alvin]
● The reagent (mumps antigens) are added to the serum
sample which promotes agglutination.
● There is also a presence of anti-IgG and RBC (came from
roosters) which act as a indicator cells
SEROLOGIC TESTING
● IMMUNOFLUORESCENCE
○ Provides rapid diagnosis
○ Detection of mumps-specific antiserum → detects
mumps virus antigens
■ As early as 2-3 days after inoculation of cell cultures
in shell vials
● ELISA
○ Detection of IgM or IgG mumps-specific antibodies
○ IgM - uniformly present early in the illness and seldom
persist longer than 60 days
○ Demonstration of mumps-specific IgM in serum drawn
early in illness strongly suggest recent infection
MOLECULAR ANALYSIS (RT-PCR)
● Preferred method
● Very sensitive
● Detects mumps genome sequences
● Identify virus strains and provides useful information in
epidemiologic studies
● Can also detect virus in many clinical samples that have
negative results in virus isolation attempts
MUMPS SKIN TEST
● Involves a delayed hypersensitivity reaction
● Determines if you had a past infection or exposure to mumps
● Similar platform to the tuberculin skin test
[PROCEDURE]:
● The mumps antigen is injected on the arm
● Injuration: the injected area of the skin will be encircled
● After 48-72 hours (2-3 days), you would go back and the
doctor would check if there is an increase in the size of
the injuration or kung lumagpas sa circle
○ (+) Increase in injuration size
■ Patient was exposed to the virus
■ There is a competent, cell-mediated immunity
SYNCYTIAL FORMATION
Syncytial formation caused by mumps virus
[SMALL DOTS]: Hemadsorption of erythrocytes onto the
surface of the cell sheet
HENDRA AND NIPAH VIRUS
OVERVIEW
● Zoonotic diseases
● Dangerous or deadly
● High mortality
● SPECIMENS: (in general)
○ Throat swabs
○ CSF for the diagnosis of encephalitis
○ Blood samples for serologic testing
MODE OF TRANSMISSION AND PATHOGENESIS
● You consider this if you have contact with animals especially
bats
○ Virus comes from the bats
● Pigs and horses are also affected
● DISEASES:
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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

○ Interstitial Pneumonia ● Isolation of Nipah virus should only be performed in a BSL-4

○ Encephalitis laboratory
● HENDRA VIRUS: affects horses in Australia ● Must be performed in the early stages of disease
● NIPAH VIRUS: affects pigs in South East Asia especially ● SPECIMENS:
Malaysia ○ Throat
○ Nasal swab
HENDRA VIRUS ○ CSF
LABORATORY DIAGNOSIS ○ Urine
○ Blood
● Isolation of Hendra virus should only be performed in a BSL-4
laboratory
● SPECIMENS:
○ Serum
○ Plasma
○ Whole blood
○ Saliva
○ Nasopharyngeal aspirate/swab
○ CSF
○ Urine
○ Tissue

● PRECAUTIONS IN COLLECTION: NIPAH INFECTION

○ Rayon or dacron-tipped swabs placed immediately in
VTM (Viral Transport Medium) after collection and stored
at 4°C

VIRAL ISOLATION
● CELL CULTURE MEDIUM:
○ Vero
○ Vero E6
○ RK13
○ MDBK
○ LLC-MK2 Active infection
○ BHK
○ HEP-2
○ HELA Cell line
○ Embryonated chicken eggs
● (+) Cytopathic Effect (CPE): large syncytia containing
multiple nuclei

SEROLOGICAL TESTS
● Enzyme Immunoassay, Microsphere Immunoassay,
Immunofluorescence Antibody
● Important considerations:
○ Serological specimen must be in an acetone-fixed
Hendra virus infected veroisic cells
Post-infection which shows an appearance of Nipah Virus in
MOLECULAR ANALYSIS Vero Cell line

● RT-PCR: greater sensitivity than conventional-gel based PCR

VIRAL ISOLATION
● Conventional PCR machines
● Vero cell line
NIPAH VIRUS ● (+) Cytopathic Effect (CPE): produces cell fusion and
LABORATORY DIAGNOSIS syncytial formation

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 UNIT 7: LABORATORY DIAGNOSIS FOR NEGATIVE-SENSE (-) ssRNA VIRUSES

5. DFA staining has a 73-95% sensitivity for Influenza A Virus

SEROLOGICAL TESTS detection.
● Can detect IgM and IgG antibodies
● ELISA:
○ Viral antigen detection (IgM and IgG)
○ Commonly used method
● Serum Neutralization Test
● Microsphere immunoassay
○ You usually have carrier particle like beads
○ The antigen or reagent is attached to the beads
○ The use of beads is for easy visualization or detection
● Rapid Diagnostic test Kits
○ For POCT

MOLECULAR ANALYSIS
● RT-PCR
● Duplex nested RT-PCR (nRT-PCR)

POST TEST QUESTIONS

1. What culture media does the measles grow well? (Primary
Monkey Kidney Media)
2. To assess parainfluenza virus infections, DNA negative test
should be confirmed using shell vial culture test (using
A549 and Mink Lung)
3. The most sensitive test for RSV detection is RT-PCR.
4. The characteristics cytopathic effect caused by mumps virus
is/are: multinucleated giant cells

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