Margie Morgan, PhD, MT(ASCP),
D(ABMM)

1.Direct Staining for Antigen
2. Enzyme Immunoassay
3. Molecular Amplification
4.Viral Cell Culture

 Direct Fluorescent antibody (DFA) stain
 Collect cells from base of fresh vesicular lesion
 Stain with DFA specific for HSV and/or VZV
 Look for fluorescent cells (virus infected) using fluorescence
microscope
 More sensitive & specific method than Tzanck prep
(DFA 80% vs. Tzanck 50%)
 Tzanck prep= Giemsa stained cells from lesion
-/examine for multinucleated giant cells of Herpes
Tzanck
Tzanck
DFA

• Enzyme immunoassay (EIA) –
 Antigen/antibody complex formed – then bound to
a color producing substrate
 Used most often for:
 Detection of non culturable viruses like Rotavirus
 Influenza A and B , & Respiratory syncytial virus (RSV)
from nasal/NP swab – point of care
• Membrane lateral flow EIA Liquid/well EIA

 Molecular Amplification (for DNA or RNA)
• Rapid/Sensitive/Specific for numerous viruses
• Exceeds sensitivity of culture/ Gold standard for:
 Respiratory viruses
 HSV and Enterovirus detection from CSF
 Culture <=20% PCR >=90%
• Tests of diagnosis not cure – can continue to shed
residual virus for 7 – 30 days after initial positive test
 Molecular quantitative assays
 CMV - Quantitative assays in transplant patients
 Hepatitis B and C detection and viral load
 HIV viral load

• Inner tube wall of tube coated with monolayer of
cells in liquid growth media
• Three types of cell lines:
 Primary cell lines – direct from animal or human organ
into culture tube , will only survive one subculture
 Rhesus monkey kidney-RMK
 Diploid – semi continuous cell lines– Can survive 20 – 50
subcultures into new vials –
 Human diploid fibroblast cells, example: MRC-5-Microbiology
Research Council 5
 Continuous cell lines – can survive continuous passage
into new vials,
 Tumor lineage, HEp-2 and HeLa

 Patient specimens inoculated into cell culture
tubes, incubated, then read under light
microscopy for “Cytopathic effect” – the effect
the virus has on the cell monolayer
• The pattern of destruction of the cell monolayer
is specific for each virus type

Spin Down Shell Vial Culture –
•A way to speed up viral cell culture
•Cell monolayer is on a coverslip
•Specimen inoculated into vial with coverslip
•Centrifuge vial to induce virus invasion into cells
•Incubate @ 35C, 24-72 hours
•Direct fluorescent antibody stain of cells – target
early virus antigens (those first formed )
Cover slip

 Viral transport media (VTM) - Hanks balanced
salt solution with antibiotics
• Also known as Universal Transport Media (UTM)
• Transport of lesions, mucous membranes and throats –
specimens collected with swab
• Cell protective = protect the cell / protect the virus
 Short term transport storage 4˚C
 Long term transport(>72hours) storage-70˚C
 VTM specimens filtered (45nm filter) to eliminate
bacteria in specimen prior to being placed onto cell
monolayer

 Most likely - HSV
 Intermediate
• Adenovirus
• Influenza A and B
• Enterovirus
 Least likely
• Respiratory Syncytial Virus (RSV)
• Cytomegalovirus (CMV)
• Varicella Zoster virus (VZV)
• Amplification preferred for these viruses due to transport
issues

 Fast (@ 24-48 hours)
• HSV
 Intermediate (5 -7 days)
• Adenovirus Enterovirus
• Influenza VZV
 Slow (10 - 14 days)
• RSV
 Slowest (10 - 21 days)
• CMV
 Amplification methods desirable for most – they
can provide results the same day

 Double stranded DNA virus
 Eight human Herpes viruses
• Herpes simplex 1
• Herpes simplex 2
• Varicella Zoster
• Epstein Barr
• Cytomegalovirus
• Human Herpes 6, 7, and 8
 Latent infection with recurrent disease is the hallmark of
the Herpes viruses
 Latency occurs within small numbers of specific kinds of
cells, the cell type is different for each Herpes virus

 Transmission: direct contact/secretions
 Latency: dorsal root ganglia
 Diseases
• Gingivostomatitis
• Herpes labialis
• Ocular
• Encephalitis
• Neonatal
• Disseminated in immune suppressed
 Therapy – Acyclovir, Valacyclovir

 Herpes simplex 1 & 2 do well in culture
• Produce CPE within 24-48 hrs
• Human diploid fibroblast cells (MRC-5)Observe for
characteristic CPE
Negative fibroblast
Cell line
HSV CPE – cell rounding
starting on the edge of the
monolayer.

 Cytology/Histology – multinucleated giant cell,
intranuclear inclusions
 Cannot differentiate
from VZV
 Amplification (PCR)
 Cell culture – HSV 1, HSV 2
 Serology – more useful for proof of past infection than
for acute diagnosis, some cross reaction with 1 and 2

 Transmission: close contact
 Latency: dorsal root ganglia
 Diseases:
• Chickenpox (varicella)
• Shingles (zoster – latent infection)
 Serious disease in immune suppressed or adult patients which
progress to pneumonia or encephalitis
 Ramsay-Hunt syndrome – facial nerve / facial paralysis
 Histology – multi-nucleated giant cells like those of
Herpes simplex
 Serology useful for immune status check
 Amplification useful for disease diagnosis
 Effective vaccine has lowered the incidence of VZV in
children

Varicella-Zoster Diagnosis
Cell culture at 5 – 7 days
Limited # of infected foci in
monolayer
Sandpaper look to the
monolayer background with
scattered rounded cells –
Diploid fibroblast cells
Young wet vesicular lesions are best
for culture and/or molecular testing
Lesions of zoster usually over one
dermatome

 Transmitted by blood transfusion , vertical and
horizontal transmission to fetus, or close contact
 Latency: Macrophages
 Disease: Infection is asymptomatic in most individuals
• Congenital – most common cause of TORCH
• Perinatal
• Immunocompromised – Primary disease most serious
 Laboratory Diagnosis:
• Cell culture CPE (Human diploid fibroblast)
• PCR and quantitative PCR (best method)
 Due to persistent shedding it is best to do quantitative PCR to detect high
viral loads, most consistent with ongoing infection
• Histopathology
 Treatment: ganciclovir, foscarnet, cidofovir

Cell culture -
CMV infected fibroblast monolayer
with grape like clusters of rounded
cells
Histopathology – Intranuclear and
Intracytoplasmic inclusions – known
as OWL EYE inclusions

 Transmission - close contact, saliva
 Latency - B lymphocytes
• Cell receptor CD21
 Diseases include:
• Infectious mononucleosis
• Lymphoreticular disease
• Oral hairy leukoplakia
• Burkitt’s lymphoma
• Nasopharyngeal Carcinoma
• 1/3 Hodgkin’s lymphoma
 Will not grow in cell culture
 Serology most used for diagnosis
 PCR techniques developed
EBV infection with B cell
transformation

 HA react with antigens phylogenetically
unrelated to the antigenic determinants
against which they were raised
 Human HAs secondary to EBV are detected by
the ability to react patient serum with horse or
cattle rbcs
• theory of the Monospot test
 HA rise in the first 2 - 3 weeks of EBV infection,
then rapidly fall at @ 4 weeks

VCA = viral capsid antibody
EBNA = Epstein Barr nuclear antigen
EA = early antigen

Anti-EBV antibodies Interpretation
VCA IgM VCA IgG EBNA-1 IgG
Negative Negative Negative No immunity
Positive Negative Negative Acute infection
Positive Positive Negative Acute infection
Negative Positive Positive Past infection
Negative Positive Negative Acute or past infection
Positive Positive Positive Late primary infection
Negative Negative Positive Past infection
VCA = viral capsid antibody
Serologic Diagnosis of EBV

 HH6
• Roseola [sixth disease]
• 6m-2yr high fever & rash
 HH8
• Kaposi’s sarcoma
• Castleman disease
• Primary effusion
lymphoma
Onion skin pattern of
Castleman disease

 DNA - non enveloped/ icosahedral virus
 Latent: lymphoid tissue
 Transmission: Respiratory and fecal-oral route
 Diseases:
• Adenovirus type 14 – virulent respiratory strain
/ pneumonia
• Pharyngitis (year round epidemics)
• Gastroenteritis in children
 Adenovirus types 40 & 41
• Kerato-conjuctivitis – red eyes for @ 2 wks
• Disseminated infection in transplant patients
• Hemorrhagic cystitis in immune suppressed

 Diagnosis
• Cell culture (CPE)
 CPE in 2-5 days with round cells connected by strands
 Grows best in Heteroploid continuous passage cell
lines (HeLA, Hep-2)
• Amplification (PCR)* superior for respiratory infection
• Histology - Intranuclear inclusions / smudge cells
• Antigen detection –
 staining respiratory cells by DFA for Respiratory
infections
 Stool antigen for 40/41 diarrhea strains
• Supportive treatment – no specific viral therapy
Round cells with
stranding

Adenovirus Smudge cells-
(Membranes become blurred)
and intranuclear inclusions

 DNA virus
 Parvovirus B19
• Erythema infectiosum (Fifth disease) – headache
rash and cold-like symptoms in the child
• In pregnant, infection in 1st
trimester, hydrops fetalis
leading to miscarriage
• Aplastic crisis in patients with chronic hemolytic
anemia and AIDS
• Histology - virus infects
mitotically active erythroid
precursor cells in bone marrow
• Molecular and Serologic methods
to aid diagnosis
Slapped face appearance
of fifth disease

Infectious and oncogenic or
potentially oncogenic DNA
viruses

 Diseases:
 Skin and anogenital warts,
 Benign head and neck tumors,
 Cervical and anal intraepithelial neoplasia and cancer
 HPV types 16, 18, & 45 = 94% Cervical CA
 HPV types 6 and 11 = 90% Genital warts
 Pap Smear for detection of HPV
 Hybrid capture DNA probe for detection and typing
 PCR* – FDA cleared platforms for detection/typing,
capable of detecting many HPV types
 Three vaccines - 1°to guard against HPV 6,11,16,18
Pap smear

• JC virus [John Cunningham]
 Progressive multifocal leukoencephalopathy -
 PML -Encephalitis of immune suppressed
 Destroys oligodendrocytes in brain
• BK virus
 Causes latent virus infection in kidney
 Progression due to immune suppression
 Hemorrhagic cystitis
• Histology/PCR to aid diagnosis
Giant Glial Cells of JCV

 Enveloped DNA – Hepadna virus
 Hepatitis B clinical disease
• 90% acute
• 1% fulminant
• 9% chronic
 Carrier state in liver can lead to cirrhosis
and hepatic cell carcinoma……. transplant
 Antiviral therapies to prevent spread
• Serology for diagnosis
• Vaccinate to prevent

 Surface Antigen Positive
• Active Hepatitis B or Chronic Carrier, if detected
 Do Hep B Quantitation
 Do Hep e antigen – if positive, Chronic carrier and worse
prognosis
 Core Antibody Positive
• Immune due to prior infection, acute infection or chronic
carrier
Surface Antibody Positive
• Immune due to prior infection or vaccine

Hepacivirus – Hepatitis C
Flavivirus – West Nile, Dengue,
Zika, andYellow Fever

 Spread parenteral - drug abuse, blood products
or organ transplants (prior to 1992), poorly
sterilized medical equipment, sexual (low risk)
 Effects only humans and chimpanzees
 Approx 3.2 mil persons in US have chronic HepC
 Seven major genotypes (1-7)
• Acute self limited disease that progresses to a
disease that mainly affects the liver
• Type 1 virus most common in USA
• Infection persists in @ 75-85%/ no symptoms
• 5 - 20 % develop cirrhosis
• 1-5 % associated with hepatocellular CA
 liver transplantation

 Diagnosis:
• Hepatitis C antibody test
 If Hep C antibody detected perform
 RNA quantitative assay for viral load
 Genotype of virus for proper therapy selection/duration
 Assessment of liver disease - ? cirhhosis
 No vaccine available
 Antivirals currently FDA cleared that can cure >=
85% of patients infected with Hepatitis C

•Dengue – “breakbone fever”
 Vector Aedes mosquito / Asia and the Pacific
 Fever, severe joint pain, rash
 Small % progress to hemorrhagic fever
•Diagnosis – Serology(IgM for acute infection)
 Zika virus
•Vector: Aedes mosquito
•Current outbreak began in South America (Brazil) and spread
to central America, Caribbean and US (Miami)
•Clinically a milder form of Dengue in most adults – but has a
neurologic tropism
 Microcephaly in fetuses borne to infected moms
 Potential developmental issues in infected children
 Guillain- Barre syndrome during ongoing Zika infection
•Diagnosis: Serum Antibody IgM and PCR serum, urine,
amniotic fluid and CSF

• West Nile
 Vector Aedes and Culex mosquito
 Common across the US,
 Bird primary reservoir, horses also at risk
 Fever, Headache, Muscle weakness, 80%
asymptomatic. Small % progress to encephalitis.
Meningitis, flaccid paralysis IgG) and PCR

 Chikungunya virus
 Vector Aedes mosquito with origin in Asia and African
continents
 Recent migration to the Caribbean and SE USA with
mosquito migration
 Travel advisory to the Caribbean
 Acute febrile illness with rash followed by extreme joint
pain, less fatalities than Dengue / no hemorrhagic
phase/ RNA virus
 When screening for ZIKA – need to rule out
infection with Dengue and Chikungunya.
Similar diseases with very different sequelae

 >20 outbreaks since discovery in 1976
• Most recent Dec 2013 - West Africa
• Prolonged outbreak due to area effected had high population
with limited medical resources
 Transmission direct contact with bodily fluids – fatality rate 55%
• Animal reservoir (?) fruit bats
 Asymptomatic are not contagious
 Fever, weakness, myalgia, headache, travel history
• Also consider malaria and typhoid in the differential
 Susceptible to hospital disinfectants
 Testing (EIA, PCR) at CDC – positive >= 4 days of illness
 RNA virus
 Level A agent of Bioterrorism

SARS - Severe Acute Respiratory Syndrome –
Outbreak in China 2003 – spread to 29 countries
Initially dry cough and/or shortness of breath with development of
pneumonia by day 7-10 of illness Lymphopenia in most cases
Laboratory testing public health laboratories (CDC) -antibody
testing enzyme immunoassay (EIA) and reverse transcription
polymerase chain reaction (RT-PCR) tests for respiratory, blood, and
stool specimens. These are RNA single strand viruses.
• MERS - Middle East Respiratory Syndrome
• Isolated mostly to Arabian peninsula (2012)
• Direct contact with infected camels
• Close human to human contact can spread infection – no
outbreaks – 30% fatality rate
• Fever, rhinorrhea, cough, malaise followed by shortness of breath

 Diverse group of > 60 viruses – SS RNA
• Infections occur most often in summer and fall
• Polio virus - paralysis
 Salk vaccine Inactive Polio Vaccine (IPV)** recommended
 Sabine vaccine Live Attenuated Vaccine (OPV)
• Coxsackie A – Herpangina – vesicular oral lesions
• Coxsackie B – Pericarditis/Myocarditis
• Enterovirus – Aseptic meningitis in children, hemorrhagic
conjunctivitis
• Echovirus – various infections, intestine
• Rhinoviruses – common cold
 Grow in cell culture * 5-7 days (Diploid mixed cell
culture – Primary Monkey Kidney)
 PCR superior for diagnosis , more rapid and sensitive for
all viruses

CPE of Enterovirus
Teardrop and kite like cells in
Rhesus Monkey Kidney cell culture
Uninfected cells

 Fecal – oral transmission, contaminated food or person to person
 80% develop symptoms – jaundice & elevated aminotransferases
 Usually – short incubation (15- 50 days), abrupt onset, low
mortality, no carrier state
 Diagnosis – serology, IgM positive in early infection to differ from
other Hepatitis viruses
 Antibody is protective and lasts for life
 Vaccine available

Influenza virus A
Influenza virus B

 Hemagglutinin and Neuraminidase glycoproteins spikes
on outside of viral capsid
• Gives Influenza A the H and N designations – such as
H1N1 and H3N2
 Antigenic drift - minor change in the amino acids of
either the H or N glycoprotein
 Cross antibody protection will still exist so an
epidemic will not occur
 Antigenic shift - genome re assortment with a “new”
virus created/usually from a bird or animal/ this could
create a potential pandemic
 H5N1 = Avian Influenza
 H1N1 = 2009 Influenza A

 Disease: fever, malaise …. Death from respiratory
complications or secondary bacterial infection
 Diagnosis
• Cell culture obsolete [RMK]
• Enzyme immunoassay (EIA) lateral flow membrane can be
used in point of care testing
• Amplification (PCR) gold standard for detection
 Treatment: Amantadine and Tamiflu (Oseltamivir)
• Seasonal variation in susceptibility but Tamiflu usually
sensitive
 Influenza B
• Milder form of Influenza like illness
• Usually <=10% of cases /year
 Vaccinate – Trivalent vaccine -2 A viruses/1 B virus

Measles
Parainfluenza 1,2,3,4
Mumps
Respiratory Syncytial Virus
Human Metapneumovirus

• Fever, Rash, Dry Cough, Runny Nose,
Sore throat, inflamed eyes (photosensitive)
 Can invade lung
• Respiratory spread - very contagious
• Koplik’s spots – bluish discoloration inner
lining of the cheek is pathognomonic
• Subacute sclerosing panencephalitis [SSPE]
 Rare chronic degenerative neurological disease
 Persistent infection with a mutated measles virus, due to
mutated virus there is total lack of an immune response
• Diagnosis: Clinical symptoms and Serology
• Vaccinate – MMR (Measles, Mumps, Rubella) vaccine
• Treatment: Nothing specific, Immune globulin, vitamin A
Measlessyncytium
H and E stain/ lung

 Types 1,2,3, and 4
 Person to person spread
 Disease:
• Upper respiratory tract infection in adults
and children with fever, runny nose and
cough
• Lower respiratory tract infection - Croup,
bronchiolitis and pneumonia more likely in
children, elderly and immune suppressed
 Heteroploid - continuous cell lines (Hep-2) for
culture – not suggested (slow and insensitive)
 PCR** methods are gold standard
 Supportive therapy only available

 Person to person contact
 Parotitis, but can also cause
infections in other sites:
Testes/ovaries, Eye, Inner ear, CNS
 Diagnosis: clinical symptoms and
serologic tests
 Prevention: MMR vaccine
 No specific therapy, supportive

 Respiratory disease - common cold to pneumonia,
bronchiolitis to croup, serious disease in infants and
immune suppressed
• Classic disease: Young infant with bronchiolitis
 Transmission by contact and respiratory droplet
 Specimen: Nasophayrngeal, nasal swab, nasal lavage
 Diagnosis: EIA (point of care), cell culture (heteroploid,
continuous cell lines), PCR is gold standard**, and lung
biopsy
 Treatment: Supportive, ribavirin
Classic CPE =
Syncytium formation
heteroploid cell line
Syncytium formation
In lung tissue

 1st
discovered in 2001 – community acquired
respiratory tract disease in the winter
• @95% of cases in children <6 years of age
• Upper respiratory tract disease
• Lower respiratory tract disease - 2nd
only to RSV in
the cause of bronchiolitis
 Will not grow in cell culture
 Amplification (PCR) for detection
• Specimen: Nasal swab or NP
 Treatment: Supportive

Rotavirus

 Winter - spring seasonality
• Gastroenteritis with vomiting and fluid loss –
most common cause of severe diarrhea in
children 6m – 2 yrs
• Fecal – oral spread
 Major cause of childhood death / 3rd
world
countries
 Diagnosis – cannot grow in cell culture
• Enzyme immunoassay, PCR
 Vaccine available
Rota = Wheel
EM Pix

Norovirus

 Spread by contaminated food and water, feces &
vomitus – takes <=20 virus particles to cause
infection – so highly contagious
 Tagged the “Cruise line virus” – numerous reported
food borne epidemics on sea aboard cruise liners
 Leading cause of epidemic gastroenteritis –
worldwide on land and sea
• Fluid loss from vomiting can be debilitating
 Disease course usually limited, 24-48 hours
 PCR for diagnosis
• Cannot be grown in cell culture

Human Immunodeficiency Virus
HIV

 CD4 primary receptor site
for entry of HIV into the lymphocyte
 Reverse transcriptase enzyme
converts genomic RNA into DNA
 Transmission - sexual, blood and blood product
exposure, perinatal
 Non infectious complications:
• Lymphoma, KS, Anal cell CA, non Hodgkins
Lymphoma

Antibody EIA with Western Blot confirmation (old way)
 Positive tests must be confirmed with a Western blot test
 Western blot detects gp160/gp120 (envelope proteins), p
24 (core), and p41(reverse transcriptase)
 Must have at least 2 solid bands on Western blot to
confirm as a positive result
New test - Antigen/antibody combination (4th generation)
immunoassay* that detects IgG and IgM HIV-1 and HIV-2
antibodies and HIV-1 p24 antigen to screen for established
and acute infection
Detects infection earlier (@ 2- 4 weeks
Positive patients on either test require additional testing:
 HIV viral load quantitation >= 100 copies
 Resistance Testing – report subtype to optimize therapy
 Most isolates in USA type B
•Monitor CD4 counts for infection severity

 Non-compliant patients or newly diagnosed
• Pneumocystis – most common in US
• Cryptococcus neoformans & Histoplasma
capsulatum (disseminated)
• TB/Mycobacterium avium complex (disseminated)
• Microsporidia and Cryptosporidium (Intestinal)
• Hepatitis B
• Hepatitis C
• STD’s – Syphilis, GC, Chlamydia
 Syphilis rate high (mucosal contact)

RNA Virus
Rubella

Known as the “Three day measles” or German measles
Rash, low grade fever, cervical lymphadenopathy
Respiratory transmission
Congenital rubella –
• occurs in a developing fetus of a pregnant women
who has contracted Rubella, highest % (50%) in the
first trimester pregnancy
• Prior to Zika it was the neurotropic virus of the fetus
• Deafness, eye abnormalities, congenital heart disease
Diagnosis - Serology in combination with clinical
symptoms
Live attenuated vaccine (MMR) to prevent

Hantavirus

 USA outbreak in the four corner states
(NM,AZ,CO,UT) on an Indian reservation in 1993
brought attention to this virus
 Source - Urine and secretions of wild field mice
• Deer mouse (picture) and cotton rat
 Myalgia, headache, cough and respiratory failure
 Found in states west of the Mississippi River
 Diagnosis by serology
 Supportive therapy

Smallpox virus (Variola virus)
Vaccinia virus

 Variola virus – agent of Smallpox
 Vaccinia virus - active constituent in the Smallpox
vaccine, it is immunologically related to smallpox,
• Vaccinia can cause disease in the immune suppressed, which
prevents vaccination of this population
• Eradication of smallpox occurred in 1977
 Disease begins as maculopapular rash and progresses to
vesicular rash -
• all lesions in same stage of development in one body area – rash
moves from central body outward
 Category A Bioterrorism agent (can maim or kill)
 Requires BSL4 laboratory (self contained lab)
 Reported to public health department for investigation

Chicken pox – Lesions in
different stage of development
Smallpox – all lesions same
stage of development
Chickenpox vs Smallpox lesions

Rabies virus

 Worldwide in animal populations
• Bat and raccoons primary reservoir in US
• Dogs in 3rd
world countries
 Post exposure shots PRIOR to the development of symptoms
prevent infection
 Rabies is a neurologic disease – classic symptom is salivation,
due to paralysis of throat muscles
 Detection of viral particles in the brain by Histologic staining
known as Negri bodies is diagnostic
 Public health department should be contacted to assist with
diagnosis

Rabies virus particles
EM showing the bullet
shaped virus
Negri bodies –
Intracytoplasmic
brain biopsy specimen

 Rare, degenerative fatal brain disorder
 Transmissible spongiform encephalopathies
(TSE) name established from the microscopic
appearance of infected brain
 Caused by type of protein - prion
 Confirmation by brain biopsy
 Safety – prevent transmission
• Universal Precautions
• Use disposable equipment
when possible
Spongiform change in the
Gray matter