Virology 2016

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

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

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

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

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

6. • Inner tube wall coated with monolayer of cells plus
liquid growth media
• Three types of cell lines:
 Primary cell lines – direct from animal or human organ
into culture tube subculture once
 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

7.  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 is specific for each
virus type

8. Spin Down Shell Vial Culture –
•Speed up virus detection
•Cell monolayer on coverslip/vial
•Specimen inoculated into vial
•Centrifuge vial to induce virus invasion into cells
•Incubate @ 35C, 24-72 hours
•Direct fluorescent antibody stain of coverslip – target
early virus antigens (those first formed )
Cover slip

9.  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

10.  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

11.  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

13.  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

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

15.  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

16.  Cytology/Histology – multinucleated giant cell,
intranuclear inclusions
 Cannot differentiate
from VZV
 Amplification (PCR)
 Serology – more useful for proof of past infection than
for acute diagnosis

17.  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
 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 incidence of VZV

18. 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

19.  Transmitted by blood transfusion , vertical and
horizontal transmission to fetus, also by close contact
 Latency: Macrophages
 Disease: Infection 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)
• Histopathology
 Treatment: ganciclovir, foscarnet, cidofovir

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

21.  Transmission - close contact, saliva
 Latency - B lymphocytes
 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
EBV infection with B cell
transformation

22.  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 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

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

24. 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 infection1
Positive Positive Positive Late primary infection
Negative Negative Positive Past infection
Serologic Diagnosis of EBV

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

27.  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 @ 2 wks
• Disseminated infection in transplant patients
• Hemorrhagic cystitis in immune suppressed

28.  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
• Stool EIA for enteric infections
• Antigen detection – staining respiratory cells by DFA
for Respiratory infections
• Supportive treatment – no specific viral therapy
Round cells with
stranding

29. Adenovirus Smudge cells-
and intranuclear inclusions

31.  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 Serology methods
to aid diagnosis
Slapped face appearance
of fifth disease

32. Infectious and oncogenic or
potentially oncogenic DNA
viruses

33.  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
 Three vaccines - 1°to guard against HPV 6,11,16,18
Pap smear

34. • JC virus [John Cunningham]
 Progressive multifocal leukoencephalopathy -
 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

36.  Enveloped DNA – Hepadna virus
 Hepatitis B clinical disease
• 90% acute
• 1% fulminant
• 9% chronic
 Carrier state can lead to cirrhosis and
hepatic cell carcinoma
 Therapies under investigation
• Serology for diagnosis
• Vaccinate to prevent

37.  Surface Antigen Positive
• Active Hepatitis B or Chronic Carrier
 Do Hep B Quantitation
 Do Hep e antigen – 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

38. Hepacivirus – Hepatitis C
Flavivirus – West Nile, Dengue,
andYellow Fever

39.  Spread parenterally - 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 USA have chronic
Hepatitis C
 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 require liver transplantation

40.  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 in clinical trials and/ or FDA
cleared that can cure >= 85% of patients infected
with Hepatitis C

41. •Dengue – “breakbone fever”
 Vector Aedes mosquito / Asia and the Pacific
 Fever, severe joint pain, rash
 Small % progress to hemorragic fever
 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
•Diagnosis – Serology(IgM, IgG) and PCR

42.  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
 Zika virus
• Vector Aedes mosquito
• Common in South America (Brazil)
• Clinically like a mild form of Dengue
• **Microcephaly in fetuses born to infected mothers
 Diagnosis
• Immunoassays for Antibody & PCR (serum and CSF)

43.  >20 outbreaks since discovery in 1976
• current outbreak Dec 2013 - West Africa
• Prolonged due to area effected is high population with limited
medical reaources
 Transmission direct contact with bodily fluids – fatality rate 55%
• Animal reservoir (?) fruit bats
 Asymptomatic are not contagious
 Fever, weakness, myalgias, headache, travel history
• Consider malaria and typhoid
 Susceptible to hospital disinfectants
 Testing (EIA, PCR) at CDC – pos >= 4 days of illness

44. SARS - Severe Acute Respiratory Syndrome –
Outbreak in China 2003 – spread to 29 countries
Initially dry cough and/or shortness of breath 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.
• MERS - Middle East Respiratory Syndome
• Isolated 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

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

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

48.  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

49. Influenza virus A
Influenza virus B

50.  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

51.  Disease: fever, malaise …. death
 Diagnosis
• Cell culture obsolete [RMK]
• Enzyme immunoassay (EIA) on paper membrane can be used
in point of care
• Amplification (PCR) gold standard for detection
 Treatment: Amantadine and Tamiflu (Oseltamivir)
• Seasonal variation in susceptibility
 Vaccinate to prevent
 Influenza B
• Milder form of Influenza like illness
• Usually <=10% of cases /year

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

53.  Measles
• Fever, Rash, Dry Cough, Runny Nose,
Sore throat, inflamed eyes (photosensitive)
 Can invade lung (see HE of Lung)
• Respiratory spread - very contagious
• Koplik’s spots – bluish discoloration inner
lining of the cheek
• Subacute sclerosing panencephalitis [SSPE]
 Rare chronic degenerative neurological disease
 Persistent infection with mutated measles virus due to
lack of immune response
• Diagnosis: Clinical symptoms and Serology
• Vaccinate – MMR (Measles, Mumps, Rubella) vaccine
• Treatment: Immune globulin, vitamin A
Measlessyncytium

54.  Types 1,2,3, and 4
 Person to person spread
 Disease:
• Upper respiratory tract infection in adults –
more serious in immune suppressed
• Croup, bronchiolitis and pneumonia in
children
 Heteroploid cell lines (Hep-2) for culture
 PCR methods are gold standard
 Supportive therapy only available

55.  Person to person contact
 Classic infection is Parotitis, but can
cause infections in other sites:
T

56.  Respiratory disease - common cold to pneumonia, bronchiolitis to
croup, serious disease in immune suppressed
• Classic disease: Young infant with bronchiolitis
 Transmission by contact and respiratory droplet
 Specimen: Naso-phayrngeal, nasal swab, nasal lavage
 Diagnosis: EIA, cell culture (heteroploid cell lines), PCR is gold
standard, lung biopsy
 Treatment: Supportive, ribavirin Histology
Classic CPE = Syncytium formation
In heteroploid cell line

57.  1st
discovered in 2001 – community acquired
respiratory tract disease in the winter
 Common in young children – but can be seen in all
age groups
• @95% of cases in children <6 years of age
• Upper 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

58. Rotavirus

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

60. Norovirus

61.  Spread by contaminated food and water, feces &
vomitus – takes <=20 virus particles to cause
infection – highly contagious
 Tagged the “Cruise line virus” – numerous reported
food borne epidemics on land and sea
 Leading cause of epidemic gastroenteritis – more
virulent GII.4 Sydney since spring 2012
• Fluid loss from vomiting can be debilitating
 Disease course usually limited, 24-48 hours
 PCR for diagnosis
• Cannot be grown in cell culture

62. Human Immunodeficiency Virus
HIV

63.  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

64. 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
 Most isolates in USA type B
•Monitor CD4 counts for infection severity

65.  Non-compliant patients or newly diagnosed
• Pneumocystis
• 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)

66. RNA Virus
Rubella

67. Known as the “Three day measles” – 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
• Deafness, eye abnormalities, congenital heart disease
Diagnosis - Serology in combination with clinical
symptoms
Live attenuated vaccine (MMR) to prevent

68. Hantavirus

69.  USA outbreak in four corner states (NM,AZ,CO,UT)
on 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
 No therapy

70. Smallpox virus (Variola virus)
Vaccinia virus

71.  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 (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

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

73. Rabies virus

74.  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 sympton 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

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

76.  Rare, degenerative fatal brain disorder
 Transmissable spongiform encephalopathies
(TSE) name established from the microscopic
appearance of brain with infection
 Caused by type of protein - prion
 Confirmation by brain biopsy
 Safety – prevent transmission
• Universal Precautions
• Use disposable equipment
when possible