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

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

3.  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

4. • 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
 Point of Care: Influenza A /B , & Respiratory syncytial virus (RSV)
• Membrane lateral flow EIA Liquid/well EIA

5. • 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 – survive continuous passage into new vials,
 Tumor lineages, HEp-2 and HeLa

6.  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

7. Spin Down Shell Vial Culture –
•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

8.  Molecular Amplification (for DNA or RNA)
• Rapid/Sensitive/Specific for numerous viruses
• Exceeds sensitivity of culture/ Standard of Practice for:
 Respiratory viruses
 Meningitis/Encephalitis
 Lesions - HSV
• 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

9.  Viral transport media (VTM) - Hanks balanced salt
solution with antibiotics, cell protective
• Also known as Universal Transport Media (UTM)
• Transport of lesions, mucous membranes, nasal & throat
specimens collected with swab
 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

11.  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
 Latency (hallmark of Herpes viruses) occurs within small
numbers of specific kinds of cells, the cell type is different
for each Herpes virus

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

13.  Herpes simplex 1 & 2
• Produce CPE within 24-48 hrs
• Human diploid fibroblast (MRC-5)
• Observe for characteristic CPE
Negative
fibroblast
Cell line
HSV CPE – cell
rounding starting on the
edge of the monolayer.
Histology/Cytology
Multinucleated Giant Cells
Cannot Differentiate from VZV
Molecular Amplification
Serology – best for past
infections

14.  Transmission: close contact
 Latency: dorsal root ganglia
 Diseases:
• Chickenpox (varicella)
• Shingles (zoster – latent infection)
 Serious disease in adults and immune suppressed with
progression to pneumonia and/or encephalitis
 Ramsay-Hunt syndrome – facial nerve / facial paralysis
 Histology – multi-nucleated giant cells
 Serology useful for immune status check
 Cell culture – growth 5-7 days, fibroblasts
 Sandpaper like appearance with rounded cells
 Amplification useful for disease diagnosis
 Effective vaccine has lowered the incidence of VZV in
children

15.  Transmitted by blood transfusion , vertical and horizontal
transmission to fetus, or close contact
 Latency: Macrophages
 Disease: Infection asymptomatic in most
• Congenital – most common cause of TORCH
• Perinatal
• Immunocompromised – 1° disease most serious
 Laboratory Diagnosis:
• Cell culture CPE (Human diploid fibroblast)
• PCR and quantitative PCR (best method)
 Due to persistent shedding - do quantitative PCR
to detect high viral loads, most consistent
with ongoing infection
• Histopathology – Intranuclear and
intracytoplasmic inclusions (Owl eye)
 Treatment: ganciclovir, foscarnet, cidofovir

16.  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 – Heterophile antibody – patient serum reacts
with horse and cattle rbcs (Monospot test)
 PCR techniques developed
EBV infection with B cell
transformation

17. 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

18.  HH6
• Roseola [sixth disease]
• 6m-2yr high fever & rash
 HH8
• (1) Kaposi’s sarcoma
• (2) Castleman disease
• (3) Primary effusion
lymphoma
Onion skin pattern
of Castleman
disease
1
1

19.  DNA - non enveloped/ icosahedral virus
 Latent: lymphoid tissue
 Transmission: Respiratory and fecal-oral route
 Diseases:
• Pharyngitis (year round epidemics)
• Pneumonia
• Gastroenteritis in children
 Adenovirus types 40 & 41
• Kerato-conjunctivitis – red eyes for @ 2 weeks
• Disseminated infection in transplant patients
• Hemorrhagic cystitis in immune suppressed

20.  Diagnosis
• Cell culture (CPE)
• Amplification (PCR)* superior for respiratory infection
• Stool antigen detection by EIA (40/41 strains)
• Supportive treatment only – no specific viral therapy
• Histology - Intranuclear inclusions / smudge cells – membranes become
blurred
Round cells 2 – 5 days
with Stranding – HeLA
or Hep-2 cells

21.  DNA virus - humans only
 Parvovirus B19
• Erythema infectiosum (Fifth disease) – headache rash and
cold-like symptoms in the child
• Hydrops fetalis - in pregnant, infection in 1st
trimester, leading
to miscarriage
• Aplastic crisis in pre-existing bone marrow stress
• Chronic hemolytic anemia HIV/AIDS
• Arthritis and Arthralgias
• Histology - virus infects
mitotically active erythroid
precursor cells in bone marrow
• Molecular and Serologic methods
to aid diagnosis
Slapped face appearance
of fifth disease

22.  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
 PCR – Detection with typing of many HPV types
 Guidelines suggest both PAP and HPV PCR for women
30 - 65 years of age / 5 years
 Three vaccines - 1°to guard against HPV 6,11,16,18
Pap smear

23. • 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

24.  Enveloped DNA virus
 Spectrum of the Hepatitis B symptomatology
• Acute phase - disease varies from subclinical hepatitis to
icteric hepatitis fulminant, acute, and subacute hepatitis
• Chronic phase - chronic hepatitis, cirrhosis, and
hepatocellular carcinoma (HCC) during the chronic phase
• Vaccinate to prevent
• Therapies available to treat
• PCR and Serology for diagnosis

25.  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

26.  Parenteral transmission - drug abuse, blood products or organ
transplants (prior to 1992), poorly sterilized medical
equipment, sexual
 Effects 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
 Can lead to liver transplantation

27.  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 - ? cirrhosis
 No vaccine available
 Antivirals currently FDA cleared that can cure >=
85% of patients infected with Hepatitis C

28. •Dengue – “breakbone fever”
•Vector Aedes mosquito / Asia and the Pacific
•Disease
 Fever, severe joint pain, rash
 Small % progress to hemorrhagic fever
•Serology -IgM for acute infection
 Zika virus
•Vector: Aedes mosquito
•2016 outbreak began in South America (Brazil) and spread to central America,
Caribbean and US (Miami)
•Clinically a milder disease than Dengue in most adults
•Neurologic tropism
 Microcephaly in fetuses borne to infected moms
 Potential developmental issues in infected children
 Guillain- Barre syndrome
•Diagnosis: Serum antibody IgM / PCR serum, urine, amniotic fluid and CSF

29.  Yellow fever
 Vector – Aedes aegypti
 Most cases mild with 3-4 days fever, headache, chills,
back pain, fatigue, nausea, vomiting
 15% cases toxic phase with jaundice due to liver
damage, hemorrhagic issues, with 20 – 50% fatality rate
 Diagnosis confirmed by serum PCR
 Outbreaks possible when large numbers of infected
mosquitoes are introduced into a heavily populated
area, endemic in Africa, Central and South America
• Large outbreak in Brazil 2018
 No specific anti-viral drugs
 Vaccine – life long immunity

30. • West Nile
 Vector Aedes and Culex mosquito
 Common across the US
 Bird primary reservoir, horses at risk
 Disease
 80% asymptomatic
 Fever, Headache, Muscle weakness
 Small % progress to encephalitis, meningitis, flaccid
paralysis
 Serology and PCR, molecular testing for West Nile has low
sensitivity, both are necessary
Cross reactions occur in serologic testing for Dengue, Zika and
West Nile virus, present diagnostic problems

31.  Powassan (POW) virus
 Transmitted to humans by infected ticks (Ixodes)
 Approximately 100 cases of POW virus disease were reported in the
United States over the past 10 years.
• Most cases have occurred in the Northeast and Great Lakes region
 Fever, headache, vomiting, weakness, confusion, seizures, and memory
loss, can cause significant swelling in the brain
 Long-term neurologic problems may occur.
 There is no specific treatment

32.  Chikungunya virus
 Vector Aedes mosquito
 Origin in Asia and African continents with recent migration to the
Caribbean and SE USA
 Acute febrile illness with rash followed by extreme joint pain,
 No hemorrhagic phase
 When screening for ZIKA – need to rule out infection with Dengue and
Chikungunya. Similar diseases with very different sequelae

33.  >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 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
 Level A agent of Bioterrorism

34. SARS - Severe Acute Respiratory Syndrome –
Outbreak in China 2003 – spread to 29 countries
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 PCR tests for respiratory,
blood, and stool specimens.
• MERS - Middle East Respiratory Syndrome
• Arabian peninsula (2012)
• Direct contact with infected camels
• Close human to human contact can spread infection –
no outbreaks – 30% fatality rate respiratory failure
• Fever, rhinorrhea, cough, malaise followed by shortness of breath

36.  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
• Coxsackie A – Herpangina – vesicular oral lesions
• Coxsackie B – Pericarditis/Myocarditis
• Enterovirus – Aseptic meningitis in children, hemorrhagic
conjunctivitis, Acute flaccid myelitis EV-D68
• Echovirus – various infections, intestine
• Rhinoviruses – common cold
 Grow in cell culture * 5-7 days
• Teardrop or kite like cells formed
 PCR superior for diagnosis

37.  Fecal – oral transmission, contaminated food or person to person
 Traveler’s disease
 Recent outbreaks in homeless populations without adequate
sanitary facilities
 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

38.  Hemagglutinin and Neuraminidase are glycoprotein 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

39.  Disease: fever, malaise …. Death from respiratory complications or
secondary bacterial infection
 Yearly H and N types dominate, most recently H1N1 and H3N2
 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 sensitive currently
 Influenza B
• Milder form of Influenza like illness
• Usually <=10% of cases /year
 Vaccinate – Trivalent vaccine -2 A viruses/1 B virus

40. Disease
• Fever, Rash, Dry Cough, Runny Nose, Sore throat,
inflamed eyes (photosensitive)
 Can invade lung
• Respiratory spread - very contagious
• Koplik’s spot – 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, PCR nasal or throat is best,
IgM serology can have false positive reactions
Vaccinate – MMR (Measles, Mumps, Rubella) vaccine
Treatment: Not specific, Immune globulin, vitamin A
H and E stain/ lung

41.  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
 PCR** methods standard of practice
 Supportive therapy

42.  Person to person contact
 Parotitis, other sites less common:
Testes/ovaries, Eye, Inner ear, CNS
 Diagnosis: clinical symptoms and
serologic tests
 Prevention: MMR vaccine
 No specific therapy, supportive

43.  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
 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

44.  1st
discovered in 2001 – community acquired respiratory
tract disease in 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
 Treatment: Supportive

45.  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 in 3rd
world countries
 Diagnosis – cannot grow in cell culture
• Enzyme immunoassay, PCR
 Vaccine available
Rota = Wheel
EM Pix

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

47.  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
 Infectious complications:
• Pneumocystis, Cryptococcal meningitis, TB and Mycobacterium
avium complex, Microsporidia, Cryptosporidium, STD’s , Hepatitis
B and C

48. 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 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

49. RNA Virus
Rubella

50. 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

51. Hantavirus

52.  1993 - Outbreak on Indian reservation in the four
corner states (NM,AZ,CO,UT) brought attention to
this virus
 Source - urine and secretions of wild deer mouse
and cotton rat
 Recent outbreak from transmission from pet rats to
humans/ Yosemite National Park cabins
 Myalgia, headache, cough and respiratory failure
 Found in states west of the Mississippi River
 Diagnosis by serology
 Supportive therapy

53. Smallpox virus (Variola virus)
Vaccinia virus

54.  Variola virus – agent of Smallpox, eradicated in 1977
 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
 Disease begins as maculopapular rash
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
 Requires BSL4 laboratory (self contained lab)
 Reported to public health department for investigation

55. Rabies virus

56.  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
progression
 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 and detection of rabies genetic material by PCR in saliva
 Public health department should be contacted to assist with diagnosis
Intracytoplasmi
Negri bodies
In brain biopsy

57.  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