Unit 4
Part 5 Viral Infections
Terry Kotrla, MS, MT(ASCP)BB
Herpes Virus Group
Include 8 viruses that cause disease.
 May result in sub-clinical infections
 Capable of establishing latent infection.
 May be reactivated under appropriate
conditions.
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Herpes Virus Group

We will discuss the following:
– Epstein-Barr virus
– Cytomegalovirus
– Herpes simplex virus type I and II
– Varicella-zoster virus
Epstein-Barr Virus (EBV)
Spread through oral transmission
 Cause of Infectious Mononucleosis.
 Other Diseases include:

– African or Burkitt’s lymphoma
– Nasopharyngeal carcinoma
– B cell lymphoma
Infectious Mononucleosis
Acute self-limiting infection of the RE
system
 Four to 7 week incubation
 Enlarged lymph nodes in the neck.
 Sore throat, fever, rash
 Malaise and extreme tiredness
 Liver and spleen involvement and
enlargement.

Infectious Mononucleosis - Lab

Hematology:
– High WBC,
– Over 20% atypical reactive lymphocytes also
known as Downey cells.
 Positive mono (heterophile antibody) test.
 Negative mono test do tests for antibodies to
EBV associated antigens.
Infectious Mononucleosis

Downey cells may be present
Heterophile Antigens/Antibodies

Heterophile antigens are a group of
similar antigens found in unrelated
animals, i.e., man, sheep, horse, dog cat,
mouse.
 Heterophile antibodies produced
against heterophile antigens of one
species will cross react with others.
Heterophile Antigens/Antibodies

Forssman antigen is an example of a
heterophile antigen and is found on the
RBCs of many species (guinea pig, dog,
cat, mouse, sheep, fowl, horse)
 Forssman antibodies formed against
Forssman antigens will agglutinate sheep
RBCs.
Paul Bunnell Test
Simple titration of sheep cell agglutinins.
 Serial dilutions of patient serum.
 Add sheep RBCs.
 Positive indicates presence of heterophile
antibodies.
 Not specific for IM, screening test only.

Davidsohn Differential
Used to determine heterophile antibody
present.
 Add patient serum to one tube with
guinea pig kidney and another with beef
red blood cells.

– Beef RBCs have IM antigen.
– Guinea pg kidney have Forssman antigen.

Harvest serum and test with sheep cells.
Davidsohn Differential
* To be considered absorbed there must be greater than a three tube
difference between the presumptive titer and the differential titer.
Heterophil Antibody
------------------------
Kidney Extract
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Beef Erythrocyte
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Infectious Mono
Not Absorbed -Aggt
Absorbed – No Aggt
Forssman
Absorbed– No Aggt
Not Absorbed-Aggt
Serum Sickness
Absorbed– No Aggt
Absorbed– No Aggt
Davidsohn Differential
Advantages
When properly
performed, this test is
specific for Infectious
Mononucleosis
 False-positive results
are rare.

Disadvantages

Davidsohn Differential test is
very time consuming and
burdensome.
Infectious Mono Slide Tests
It was discovered that horse RBCs possess
antigens which react with the antibody
associated with IM.
 Patient serum mixed with horse RBCs,
agglutination is positive.
 Latex agglutination, coat particles with
EBV antigens.

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Not diagnostic, must look at total clinical
picture.
EBV Antigens
Early antigen (EA)
 Capsid antigen (VCA)
 Membrane antigen (MA)
 Nuclear antigen (EBNA)

EBV Specific Antibodies
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EBV specific antibodies may be measured.
Pattern of appearance of EBV antigens.
Most valuable is IgM antibody to viral capsid
antigen (VCA), indicates a current infection (best
marker), lasts about 12 weeks.
Can also detect anti-early antigen (EA) (recent
infection) and anti EB nuclear antigen (EBNA)
(older infection).
ELISA and IFA most commonly used
EBV Antibody Response
Stage of
Disease
Acute
Anti-VCA
Heterophile
Antibody
Pos or Neg
Past infection Neg
IgM IgG Anti-EBNA
Pos Pos Neg
Neg Pos Pos
African or Burkitt’s Lymphoma
• EBV has been strongly implicated
• Malignant B-cell neoplasm
– presents as rapidly growing tumor of the jaw,
face or eye
– grows very quickly, and without treatment
most children die within a few months
Rare in US, equatorial Africa.
 Also associated with HIV infection.

African or Burkitt’s Lymphoma

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Although BL is a very rapidly growing tumour it
responds well to treatment.
Three pictures: before treatment, 3 days and 6
days after treatment
Nasopharyngeal Carcinoma
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Endemic in South China, Africa, Arctic Eskimos
This is a malignant tumour of the squamous
epithelium of the nasopharynx.
100% contain EBV DNA
Rates are less than 1 per 100,000 in most
populations
Nasopharyngeal carcinomas are found in
association with reactivation of latent EpsteinBarr Virus.
The exact mechanisms of association are
unknown
B-Cell Lymphoma
In most individuals infected with EBV, the virus
is present in the B-cells, which are normally
controlled by T-lymphocytes
 When T-cell deficiency exists, one clone of EBVinfected B-lymphocytes escapes immune
surveillance to become autonomously
proliferating.
 EBV induced B cell lymphomas are most
prevalent in immunocompromised patients.

Oral Hairy Cell Leukoplakia
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HIV infection stimulates reactivation of pre-existing,
latent EBV infection.
Causes viral infection of the oral cavity.
Indicator of HIV infection as well as of a person's
lessening or weakening immunity
Cytomegalovirus
Human Herpesvirus 5 (HHV-5)
 Transmission occurs from person to
person.
 Close intimate contact

– Sexual contact
– Perinatally
– Breast milk
– Organ transplant
– Blood transfusion
CMV Clinical course
Symptoms resemble IM
 In babies may cause life threatening
illness
 Patients with deficient immune systems
 AIDS patients
 Transplant patients

Cytomegalovirus
Perinatal transmission occurs in 10%.
 If infected may cause multitude of symptoms
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Petechiae
Jaundice
Hepatosplenomegaly
Neurological abnormalities
Moratlity rate 5 percent
 Survivors may exhibit hearing loss, visual
impairment and mental retardation.

CMV Immunologic response
Test for CMV antibody using paired serum
samples
 IgM antibodies produced against early and
intermediate-early (IE) CMV antigens, last
for 3 to 4 months.
 IgG appear shortly after and peak at 2 to
3 months.

CMV Laboratory Diagnosis
Range from culture and cytologic
techniques to DNA probes, PCR and
serologic techniques.
 Detection of antibodies indicator of recent
or active infection.
 Viral cultures

Microscopic examination of biopsy
specimens
CMV Lab Diagnosis
Detection of CMV antigen in cells using
IFA
 ELISA to detect antibody to CMV
 Other

– fluorescence assays,
– indirect hemagglutination, and
– latex agglutination

False positives can occur due to RA and
Epstein-Barr antibodies
Herpes Simplex Virus (HSV)
Most exposed in childhood
 Possesses viral latency – hibernation
 Two types: HSV-1 and HSV-2

HSV-1
Transmitted from person to person by
saliva or direct contact.
 Cold sores around the mouth most
common.
 Reactivation - may have several episodes
of cold sores during a lifetime

HSV-1

Symptoms
– tingling
– Numbness
– Itching
Blister forms, breaks, crusts over
 Reactivation usually caused by stress.
 Conjunctivitis, keratitis and herpetic
whitlow may occur.
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HSV-2
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Results in Herpes genitalis - lesions BELOW the waist.
Transmitted intimate sexual contact or perinatally.
Symptoms
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Pain
Tenderness
Itch
Fever
Headache
Lymphadenopathy
Malaise
HSV-2
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Blisters appear
– Males – penis
– Females – vagina and cervix
– Both – thighs buttocks
Painful, lasts 1-3 weeks
 Virus lies dormant in nearby nerves and
reactivated.

HSV – 2
Can be fatal in infants
 Woman with active infection needs Csection.
 Infants with localized infections have 70%
mortality rate
 Disseminated neonatal herpes most lethal
form.

Neonatal Herpes
Laboratory Testing for HSV
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Recovery of virus from culture
Direct examination of cells from lesion using IF
or immunoperoxidase stain
DNA probes
ELISA
Latex agglutination
RIA
Indirect IF
Serology NOT very useful
Varicella-Zoster Virus

Two different manifestations of the same
virus.
– Varicella is the primary infection, causes
chicken pox
– Herpes Zoster causes shingles and is due
to reactivation of the latent virus
Varicella
Fever and vesicular exanthema
 Small, itchy blisters surrounded by
inflamed skin.
 Begins as one or two lesions and spreads.
 Number of lesions vary greatly.
 Blister dries out and forms a scab.

Chicken Pox
Chicken Pox

Secondary complications due to infection
most common.
– May also result in pneumonia, encephalitis
and hepatitis.
– Very serious for immunocompromised children

Vaccine now available
Shingles
Chicken pox – virus goes latent
 Reactivated later in life

– Weakened immune system
– Aging
– Other factors
Shingles
The typical rash of shingles begins as
redness(erythema) followed by the
appearance of blisters.
 Eruptions follow the path of an infected
nerve.
 The trunk is the area affected in 50% to
60% of cases.
 Skin may be extremely sensitive to touch

Shingles
Shingles
Shingles
VZV Laboratory Testing

Important to distinguish VZV from other
infections
– PCR
– Direct Fluorescent Antibody staining
– Viral culture
– IgG and IgM antibody test by ELISA
Rubella Virus
RNA virus with 3 major structural proteins,
E1, E2, and C.
 Incubation 2- 3 weeks
 Highly contagious, spread through
respiratory tract.
 Causes German measles

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Rubella vaccine has resulted in 99%
decline in infections.
Rubella Symptoms

Mild and difficult to notice
– Mild fever
– Headache
– Stuffy or runny nose
– Red eyes
– Enlarged and tender lymph nodes
– Pink rash begins on face, spreads to trunk
then arms and legs, disappears in same order.
– Aching joints
Rubella
Congenital Rubella
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Congenital Rubella Syndrome most serious.
Fetus infected during first trimester.
Result in miscarriage or stillbirth,
Live-born serious birth defects or dying.
20% of the children born after such an infection
suffer the severe congenital abnormalities
10-20% of these children die within the first
year of life.
Rubella vaccine contraindicated during
pregnancy.
Rubella Syndrome
Rubella Lab testing
IgG and IgM antibodies may form at same
time
 IgM antibodies persist for 4 to 5 weeks,
IgG for life.
 Performed primarily for diagnosis of
acquired infections and to determine
immune status of pregnant patients.
 Some tests detect IgG antibodies, other
IgM.
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Rubella Laboratory Testing
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Methods include:
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hemagglutination inhibition
passive hemagglutination
neutralization
hemolysis in gel
complement fixation
fluorescence immunoassay
RIA
ELISA
latex agglutination.
Rubeola
Single stranded RNA virus best known for
its typical skin rash
 Primarily respiratory infection
 Incubation approximately 10 days, ranges
from 8-13.
 Rash appears at about day 14.
 Airborne precautions

Rubeola

Symptoms include
– Irritability
– Runny nose
– Eyes that are red and sensitive to light,
– Hacking cough, and
– High fever
– Swollen lymph nodes
Rubeola
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Fever peaks with the appearance of the rash.
Red spots with tiny grayish white heads appear
on inside of cheeks at back of mouth.
After 18 hours spots disappear and rash
develops, typically begins on forehead, then
spreads downward over face, neck, and body.
Rash appears on face first and consists of large
flat red to brown blotches that often flow into
one another.
Rash fades in the same order that it appeared
Rubeola
Rubeola Complications
Croup
 Bronchitis
 Bronchiolitis
 Pneumonia
 Conjunctivitis
 Myocarditis
 Hepatitis
 Encephalitis

Rubeola
More susceptible to ear infections or
pneumonias.
 Disease can be severe, with
bronchopneumonia or brain inflammation
 May lead to death in approximately 2 of
every 1,000 cases.
 Most severe in adults.

Measles vaccine
Live attenuated-reduce virulence of
organism but it is till alive.
 DO NOT give to:

– pregnant women,
– persons with active tuberculosis,
– leukemia,
– lymphoma,
– depressed immune systems.
– People with egg allergies
Measles vaccine
Occasionally causes side effects in persons
with no underlying health problems,
 In about 10% of cases there is a fever
between 5 and 12 days after vaccination,
 In about 5% of cases there is a rash.

Rubeola Laboratory Testing
Serology testing provides best means of
confirming a measles diagnosis
 Methods to detect rubeola antibodies
include:

– hemagglutination inhibition
– endpoint neutralization
– complement fixation
– IFA
– ELISA
Laboratory Testing
Diagnosis confirmed by presence of
Rubeola specific IgM antibodies antibodies
or four-fold rise in IgG antibody titer in
paired samples taken after rash to 10 to
30 days later.
 IgM test highly depended on time of
sample collection with 3-11 days after rash
being optimal.
 IgM false positive due to RA.

Mumps
Single stranded RNA virus.
 Mumps is transmitted by direct contact
with saliva and discharges from the nose
and throat
 iIncubation 16-18 days.
 Virus can infect many parts of the body,
especially the parotid salivary glands.

Mumps
Glands usually become increasingly
swollen and painful over a period of 1 to 3
days
 Pain gets worse
 Both the left and right parotid glands may
be affected

Mumps
Mumps
Mumps - Complications
Inflammation and swelling of the brain
 Mumps in adolescent and adult males may
also result in the development of orchitis
 May affect the pancreas or, in females, the
ovaries
 Infection in pregnant women may result in
increased risk for fetal death

Laboratory Testing
complement fixation
 hemagglutination inhibition
 hemolysis-in-gel
 neutralization assys
 IFA and
 ELISA

Laboratory Testing
Current or recent infections indicated by presence of
specific IgM antibody in single sample which can be
detected within 5 days of illness.
 Fourfold rise in specific IgG antibody in 2 samples
collected during acute and convalescent phases
 Fluorescent antibody staining for mumps antigens
 Cross-reactivity between antibodies to mumps and
parainfluenza viruses has been reported in tests for IgG,
not a problem since symptoms differ.
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The End