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SECTION 1
Morphological Diagnosis
of Viral Infections
DNA Viruses
Adenovirus Infection I-1-2
Cytomegalovirus (HHV-5) Infections I-1-6
Epstein-Barr Virus (HHV-4) Infection I-1-10
Hepatitis B and D Virus Infections I-1-12
Herpes Simplex Virus 1 and 2 (HSV-1, HSV-2) Infections I-1-16
Kaposi Sarcoma-Associated Herpesvirus (HHV-8) Infection I-1-20
Measles Virus Infection I-1-24
Parvovirus B19 Infection I-1-26
Polyomavirus Infections I-1-28
Pox Virus Infections I-1-34
Varicella-Zoster Virus (HHV-3) Infections I-1-38
Human Papillomavirus Infection I-1-40
RNA Viruses
Rabies Virus Infection I-1-50
Respiratory Syncytial Virus Infection I-1-52
ADENOVIRUS INFECTION
An atypical smudged nucleus in the lung of
an immunosuppressed patient is shown with minimal
inflammation. Immunohistochemistry demonstrated
Viral Infections: Morphological Diagnosis of Viral Infections
multiple cells positive for adenovirus.
TERMINOLOGY
Abbreviations
Adenovirus [serovar #] = Ad#
Example: Adenovirus serovar 7 = Ad7
Synonyms
The common "cold" (accounts for only 5%)
Definitions
"Adenovirus" derived from "adeno" (for adenoids
where 1st isolated)
ETIOLOGY/PATHOGENESIS
Infectious Agents
Adenoviridae family: Nonenveloped icosahedral DNA
viruses present in most vertebrates
Ubiquitous virus able to infect many cell types
Commonly found in respiratory tract and stool of
children
Initial infection
Enters epithelial cell
Replicates through entire lytic cycle
Produces cytolysis
inflammatory response (depending on patient)
Chronic infection
Unknown process occurring in various lymphoidal
tissues
Oncogenic in animal models but not, to date, in
humans
Common tool for vaccine vectors with wellcharacterized genome
Widespread use in developing novel cancer therapies
using adenovirus as vectors
I
Abnormal nuclei (smudged to Cowdry-like) are
shown adjacent to necrotic liver with no inflammation
(fulminant adenovirus hepatitis), resulting in death in an
immunosuppressed patient.
CLINICAL ISSUES
Epidemiology
Ubiquitous viral family affecting all age groups
Most adults have seropositivity to several serotypes
Immunocompetent individuals have mild, limited
disease
Site
Conjunctivitis &/or tonsillitis (Ad3, 4, 7)
Bronchiolitis &/or pneumonia
Laryngotracheobronchitis (Ad1, 2, 3, 5, 6, 7, 14)
Myocarditis (chronic leading to cardiomyopathy)
Gastroenteritis, colitis, appendicitis
Hepatitis (Ad1, 2, 5): Fulminant, similar to herpesvirus
Meningitis/encephalitis
Nephritis, hemorrhagic cystitis (rare)
Presentation
A range of clinical infections (mild to life threatening)
Primary infection (2-14 days incubation) or
reactivation of latency
Immunocompromised (all age groups) at highest risk
Mortality 50-80% in immunosuppressed children
Mortality up to 70% in immunosuppressed adults
Solid organ and bone marrow transplant recipients
Meningoencephalitis and pneumonitis increase risk in
immunocompetent population
Laboratory Tests
Serology
Challenging to interpret unless serovar matched to
symptoms
Culture from direct tissue sample > blood
Antigen detection: Stool for enteric (Ad40, Ad41)
Direct fluorescence antigen detection &/or PCR on
nasal swabs
PCR on CSF or stool
1
2
ADENOVIRUS INFECTION
Etiology
Commonly found in respiratory tract and stools of
children
Clinical Issues
Severe, life-threatening disease in immunosuppressed
High mortality in all age groups with
immunosuppression (regardless of site of infection)
Solid organ, bone marrow transplant = high suspicion
New-onset severe disease in immunosuppressed
should be rushed
Hepatitis (Ad1, 2, 5): Fulminant, similar to
herpesvirus
Microscopic Pathology
Suspicious "viral cytopathic effect" should warrant
immunohistochemistry
Key Facts
Morphological confirmation = intravenous treatment
Classically, glassy nucleus to smudged appearance
Ancillary Tests
Direct culture of tissue specimen
Immunohistochemistry
Molecular testing of tissue specimen
Top Differential Diagnoses
Herpesvirus family (all sites)
Polyomavirus (in CNS and kidney/bladder)
Diagnostic Checklist
Necrosis = fulminant disease
Low threshold for ordering confirmatory test (IHC or
PCR)
Immunohistochemistry, PCR, &/or culture confirm
diagnosis
Viral Infections: Morphological Diagnosis of Viral Infections
Natural History
Self-limited (immunocompetent)
Severe (immunosuppressed)
Treatment
Most commonly mild, self-limited disease
Severe disease = intravenous antivirals
Ribavirin, cidofovir
No randomized clinical trials to date (compassionate
use in diagnosis-proven disease)
Prognosis
Most serovars: Self-limited infection
Adenovirus serotype 14: < 1% mortality
Immunocompromised hosts
Highest mortality group
Serovar specificity to site of infection
Rising viral load = risk of severe disease
MICROBIOLOGY
Virus Features
Nonenveloped, lytic, double-stranded DNA virus
70-90 nm in size with icosahedral capsid
57 human serotypes divided into 7 species (groups AG)
26,000 to 46,000 bp genome
Culture
Grows in cell culture
HEK
HEp-2
A549 (laboratory diagnosis)
Graham 293 (enteric adenovirus)
1-3 weeks for growth
Confirmed by cytopathic effect with positive direct
antigen visualization
MICROSCOPIC PATHOLOGY
Histologic Features
Nonspecific "viral cytopathic effect" common
Classically, glassy nucleus to smudged appearance
Must differentiate from reactive cells
Selected specific tissue patterns of injury (in addition
to viral cytopathic effect)
Central nervous system
Minimal inflammation to perivascular cuffing and
increased cellularity
Necrosis is not usually a feature
Pulmonary
Minimal inflammation to diffuse alveolar damage
Necrosis is an uncommon feature
Cardiac
Limited mononuclear to dense lymphohistiocytic
intersitial inflammation with necrosis
Hepatic
Focal to extensive necrosis with limited
inflammation
Cytologic Features
Medium to large cells with variable nuclear changes
Nucleoli
Smudged chromatin
Background milieu may show necrosis or limited
inflammation
ANCILLARY TESTS
Immunohistochemistry
For adenovirus antigen(s)
Direct visualization of virus proteins
Should always be considered in concert with
herpesviruses without prior diagnosis
Example A: Patient with known adenovirus culture
or PCR positive = single IHC for adenovirus
Example B: Patient with new respiratory illness and
suspicious viral cytopathic effects on biopsy = panel
of adenovirus plus herpesvirus
I
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ADENOVIRUS INFECTION
Immunofluorescence
For adenovirus antigen(s)
Direct visualization of virus proteins
Performed on clinical nasal swabs (direct) for rapid
diagnosis
Performed on fresh frozen or FFPE tissue sections for
confirmatory diagnosis
In Situ Hybridization
Specific DNA probes for adenovirus target(s)
Available in reference laboratories
Should be used with caution due to ubiquitous
nature of virus
PCR
Molecular detection of adenoviral DNA or RNA
Most commonly used for enteric infection &/or CNS
infection
Broad panels available for screening &/or diagnosis
Qualitative or quantitative
Viral Infections: Morphological Diagnosis of Viral Infections
Directly on tissue sample, fluid, or blood
Available via reference laboratories
Should be used with caution due to ubiquitous
nature of virus
Electron Microscopy
Transmission
Demonstration of icosahedral particles consistent
with adenovirus
DIFFERENTIAL DIAGNOSIS
Bacterial Infection (Lung and Liver)
Destructive, necrotic lesions
Demonstration of bacteria on Gram or silver staining
Positive culture of bacteria from sample
Diffuse Alveolar Damage (Lung)
Patients with acute respiratory distress syndrome
(ARDS)
Any condition leading to DAD can mimic adenoviral
infection (and vice versa)
Reactive, degenerative cells may be suspicious for viral
cytopathic effect
Clinical history, culture, and immunohistochemistry
can exclude DAD of other causes
DIAGNOSTIC CHECKLIST
Clinically Relevant Pathologic Features
Suspected viral cytopathic effect should be
thoroughly evaluated due to broad differential in
immunosuppressed populations
Tissue necrosis = fulminant disease = worst prognosis
Pathologic Interpretation Pearls
In immunosuppressed, apply low threshold for
suspicious "viral cytopathic effect"
Immunohistochemistry, immunofluorescence, &/or
culture confirm diagnosis
In situ hybridization and PCR are valuable but require
caution for interpretation due to ubiquitous nature of
virus
Herpesvirus Family (All Sites)
Herpes simplex 1 & 2
Multinucleation
Classic Cowdry type A nuclei
Cytomegalovirus
2-4x size of adjacent cells
Classic Cowdry type A nuclei
Varicella-zoster virus
Classic Cowdry type A nuclei
When "viral cytopathic effect" is suspected in
immunosuppressed patients, a panel of HSV, VZV,
CMV, and adenovirus immunohistochemistry is
advised
Polyomavirus (Cerebral Cortex, KidneyBladder)
Demyelinating (polyoma) vs. meningoencephalitis
(adenovirus)
Immunohistochemistry for both agents is
confirmatory
Other Viral Infections (All Sites)
RNA viruses and others resulting in necrosis without
inflammation
Exclude adenovirus by immunohistochemistry
Reference tissue testing for confirmation of other viral
I
infections is recommended
SELECTED REFERENCES
1. Cheng X et al: Cell-cycle arrest at G2/M and proliferation
inhibition by adenovirus-expressed mitofusin-2 gene
in human colorectal cancer cell lines. Neoplasma.
60(6):620-6, 2013
2. Shauer A et al: Acute viral myocarditis: current concepts in
diagnosis and treatment. Isr Med Assoc J. 15(3):180-5, 2013
3. Tsamis KI et al: Combination treatment for glioblastoma
cells with tumor necrosis factor-related apoptosis-inducing
ligand and oncolytic adenovirus delta-24. Cancer Invest.
31(9):630-8, 2013
4. Lamps LW: Infectious causes of appendicitis. Infect Dis
Clin North Am. 24(4):995-1018, ix-x, 2010
5.
Echavarra M: Adenoviruses in immunocompromised
hosts. Clin Microbiol Rev. 21(4):704-15, 2008
6. Wong S et al: Detection of a broad range of human
adenoviruses in respiratory tract samples using a sensitive
multiplex real-time PCR assay. J Med Virol. 80(5):856-65,
2008
7. Dubberke ER et al: Acute meningoencephalitis caused by
adenovirus serotype 26. J Neurovirol. 12(3):235-40, 2006
8. Ljungman P et al: Cidofovir for adenovirus infections
after allogeneic hematopoietic stem cell transplantation:
a survey by the Infectious Diseases Working Party of the
European Group for Blood and Marrow Transplantation.
Bone Marrow Transplant. 31(6):481-6, 2003
9. Gavin PJ et al: Intravenous ribavirin treatment for severe
adenovirus disease in immunocompromised children.
Pediatrics. 110(1 Pt 1):e9, 2002
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Microscopic Features
A
p
f
p
Viral Infections: Morphological Diagnosis of Viral Infections
ADENOVIRUS INFECTION
(Left) Adenovirus infection
of the central nervous
system is presented with
neurons of the cerebral
cortex showing dark
blue/purple smudged
nuclei, red cytoplasm,
and minimal reaction or
inflammation. (Right)
denovirus-infected cells
are scattered throughout
the cerebral cortex and
spinal cord in CNS cases.
Specific involvement of
anterior horn cells is seen
here.
(Left) A low-power view
of severe pneumonia in
an immunocompromised
atient with adenovirus
infection demonstrates
diffuse alveolar damage. At
this level, the features are
consistent with DAD but
not specific to adenovirus.
(Right) High magnification o
adenovirus-induced diffuse
alveolar damage shows
viral cytopathic effect in
several cells. As with any
case of DAD, a cause may
not be identified but viral
cytopathic change should be
carefully scrutinized.
(Left) Immunohistochemistry
shows predominantly
nuclear staining with
adenovirus antibodies in
a fulminant hepatitis. The
response to adenovirus is
variable (and may be only
necrosis) but suspicious viral
cytopathic effect should be
carefully scrutinized. (Right)
Immunohistochemistry
for adenovirus antigens
shows both nuclear and
cytoplasmic staining in
neumonia with diffuse
alveolar damage.
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CYTOMEGALOVIRUS (HHV-5) INFECTIONS
Two CMV-infected cells are shown in an alveolus from
a patient with severe CMV pneumonia. Note the purple
granules in the cytoplasm of one cell and the "owl’s-
Viral Infections: Morphological Diagnosis of Viral Infections
eye" nucleus of the other.
TERMINOLOGY
Abbreviations
Cytomegalovirus (CMV)
Human herpesvirus-5 (HHV-5)
Definitions
"Cytomegalovirus" derived from "cyto-" (cell) and
"megalo-" (large)
"Herpesvirus" derived from "herpein" (to creep)
Herpesviridae: Ubiquitous enveloped icosahedral
double-stranded DNA virus present in most vertebrates
ETIOLOGY/PATHOGENESIS
Infectious Agents
Ubiquitous virus able to infect many cell types
Commonly found in saliva, urine, semen > > > other
body fluids
Initial infection
Route of entry is salivary glands
Lytic infection that is self limited
Chronic infection
Asymptomatic latency in immunocompetent
Viral shedding in infected hosts
Loss of cell-mediated immunity
(immunosuppression)
Viral reactivation and lytic cycle infection
No direct evidence of oncogenesis but may work
through oncomodulation to increase malignancy of
tumors
CLINICAL ISSUES
Epidemiology
I
Ubiquitous virus affecting all age groups
Seropositivity rates depend on population and age
~ 40% of population on average worldwide
1
CMV-infected cells within the submucosa of a severe
CMV colitis are shown with admixed cellular debris
and minimal cellular inflammatory response.
~ 60% of adults in United States
~ 90% of male homosexuals
36% in children rises to 91% of adults > 80 years
Immunocompetent individuals have asymptomatic
initial infection or (rarely) mononucleosis-like illness
Transplant patients (20-60%) experience symptomatic
infections
Site
Symptomatic infection in immunocompetent (rare)
Pneumonia
Hepatitis
Colitis
Encephalitis
Rare sites of clinical infection
Esophagus
Salivary glands
Kidneys
Adrenals
Pancreas
Mononucleosis-like initial presentation
Lymphadenopathy
Splenomegaly
Hepatitis
Mimics Epstein-Barr virus infection (mononucleosis)
Symptomatic infection in the immunosuppressed
Can involve any organ
Can involve many cell types
Presentation
A range of clinical infections (mild to life threatening)
Primary infection (9-60 days incubation) or
reactivation of latency
Immunocompetent
In utero: TORCH organism
Children and young adults: Mononucleosis-like
illness
Immunosuppressed
HIV infection
Low CD4 count
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CYTOMEGALOVIRUS (HHV-5) INFECTIONS
Viral Infections: Morphological Diagnosis of Viral Infections
Etiology
Asymptomatic latency in immunocompetent
Viral shedding in infected hosts
Loss of cell-mediated immunity
(immunosuppression)
Clinical Issues
Seropositivity rates depend on population and age
Transplant patients (20-60%) experience
symptomatic infections
Immunocompetent
In utero: TORCH organism
Solid organ transplants
Donor positive/recipient negative = highest risk
Bone marrow transplants
Donor negative/recipient positive = highest risk
Quantitative PCR on blood
Coinfection with other opportunistic pathogen
Solid organ transplants
Donor positive/recipient negative = highest risk
Bone marrow transplants
Donor negative/recipient positive = highest risk
Organ-specific symptoms
Laboratory Tests
Serology (IgG &/or IgM)
Direct or indirect immunofluorescence for CMV
antigens (urine, saliva, blood, fluids)
Culture from direct tissue sample > blood
Qualitative PCR on blood
Quantitative PCR on blood
Used to follow immunosuppression
Correlates with level of immunosuppression directly
Does not correlate with organ-specific disease
Example A: Patient with low CMV viral load may
present with severe CMV pneumonia
Example B: Patient with high CMV viral load has no
organ-specific symptoms or actual pathology
Natural History
Self-limited in immunocompetent
Moderate to severe in immunosuppressed
Moderate to severe organ-specific disease
Severe disseminated disease
Treatment
Prevention in immunosuppressed (prophylaxis)
Valganciclovir (favored)
Ganciclovir
Treatment in immunosuppressed (all sites)
Ganciclovir (first-line)
Foscarnet, cidofovir, maribavir (second-line)
CMV immune globulin
With ganciclovir for lung transplant patients
Treatment of CMV retinitis
Valganciclovir
Prognosis
Dependent on host and disease context
Key Facts
CMV organ-specific disease in immunosuppressed
HIV-infected patients
Microscopic Pathology
3-4x size of macrophages
Large "owl’s-eye" nucleus
Abundant cytoplasm distinctive red-purple
granules
Top Differential Diagnoses
Herpesvirus family
Adenovirus
Diffuse alveolar damage
TORCH: Pregnancy loss to severe long-term
complications
CMV organ-specific disease in immunosuppressed
HIV-infected patients
MICROBIOLOGY
Virus Features
Enveloped, lytic, double-stranded DNA virus
150-200 nm in size with icosahedral capsid
Human CMV (single type)
235,645 bp genome (largest of the herpesviruses)
Culture
Grows in cell culture
1-2 weeks for growth
Confirmed by cytopathic effect with positive direct
antigen visualization
Shell-vial culture
MICROSCOPIC PATHOLOGY
Histologic Features
Very large cells
Large "owl’s-eye" nucleus
Abundant cytoplasm distinctive red-purple granules
Selected specific tissue patterns of injury (in addition
to viral cytopathic effect)
Does not correlate with organ-specific disease
Unmonitored, acute-onset disease, worse prognosis
Untreated, acute-onset disease, worst prognosis
Red-purple inclusion within large cleared nucleus
With preemptive &/or prophylaxis = better
prognosis
Unmonitored, acute onset disease = worse prognosis
On highly active anti-retroviral therapy (HAART)
disease less common, best prognosis
Untreated, acute-onset disease, worst prognosis
Human fetal fibroblasts (HFF) cell lines
Rapid, pre-"viral cytopathic effect" detection by
antibody
3-4x size of macrophages
Red-purple inclusion within large cleared nucleus
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CYTOMEGALOVIRUS (HHV-5) INFECTIONS
Ischemia &/or necrosis (with vascular endothelial
infection)
Ulceration (gastrointestinal tract)
Cytologic Features
Markedly enlarged mononuclear cells
Basophilic intranuclear inclusions
Small granular cytoplasmic inclusions
ANCILLARY TESTS
Immunohistochemistry
For CMV antigen(s)
Should always be considered in concert with
herpesvirus without prior diagnosis
Example A: Patient with known positive CMV viral
load = single IHC for confirmation of suspicious cells
Example B: Patient with new-onset organ specific
symptoms and suspicious (but not diagnostic) viral
Viral Infections: Morphological Diagnosis of Viral Infections
cytopathic effects on biopsy = panel of CMV and
other herpesviruses
Immunofluorescence
Direct or indirect immunofluorescence for CMV
antigens (tissue biopsies, cytology samples)
Immunohistochemistry is preferred over
immunofluorescence for routine confirmation
Adenovirus
Suspicious viral cytopathic effect
Other Viral Infections (All Sites)
RNA viruses resulting in necrosis inflammation
Correlated with CMV immune status, viral load, and
immunohistochemistry
Reference testing for confirmation of other viral
infections is recommended
Bacterial Infections (All Sites)
Destructive, necrotic lesions
Demonstration of bacteria on Gram or silver staining
Positive cultures of bacteria from sample
Diffuse Alveolar Damage (Lung)
Patients with acute respiratory distress syndrome
(ARDS)
Any condition leading to DAD can mimic CMV
infection (and vice versa)
CMV can lead directly to DAD
Reactive, degenerative cells may be suspicious for viral
cytopathic effect
CMV can reactivate in damaged lung tissue
incidentally and not be the root cause of DAD
Clinical history, culture, and immunohistochemistry
can exclude DAD of other causes
In Situ Hybridization
Specific DNA probes for cytomegalovirus target(s)
Available in reference/research laboratories
Directly performed on tissue samples (rarely needed)
PCR
Molecular detection of CMV DNA or RNA
Most commonly used on blood to monitor
immunosuppression
Qualitative and quantitative
Directly on tissue sample, fluid, or blood (rarely
needed)
Available via reference/research laboratories
DIAGNOSTIC CHECKLIST
Clinically Relevant Pathologic Features
Suspected viral cytopathic effect should be
thoroughly evaluated due to broad differential in
immunosuppressed populations
Tissue necrosis = fulminant disease = worst prognosis
Pathologic Interpretation Pearls
In immunosuppressed, apply low threshold for
suspicious viral cytopathic effect (HIV)
Immunohistochemistry, &/or culture confirm
diagnosis
Electron Microscopy
Transmission
Icosahedral viral capsid with (infective particle)
or without (incomplete particle) surrounding cell
membrane
DIFFERENTIAL DIAGNOSIS
Herpesvirus Family (All Sites)
Herpes simplex 1 & 2
Multinucleation
Classic Cowdry type A nuclei
Varicella-Zoster virus
Classic Cowdry type A nuclei
When viral cytopathic effect is suspected,
panel of HSV, VZV, CMV, and adenovirus
immunohistochemistry is advised
SELECTED REFERENCES
1. Fishman JA: Overview: cytomegalovirus and the
herpesviruses in transplantation. Am J Transplant. 13 Suppl
3:1-8; quiz 8, 2013
2. Michaelis M et al: Oncomodulation by human
cytomegalovirus: novel clinical findings open new roads.
Med Microbiol Immunol. 200(1):1-5, 2011
3. Li YT et al: Extensive human cytomegalovirus (HCMV)
genomic DNA in the renal tubular epithelium early after
renal transplantation: Relationship with HCMV DNAemia
and long-term graft function. J Med Virol. 82(1):85-93,
2010
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Microscopic Features
p
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Viral Infections: Morphological Diagnosis of Viral Infections
CYTOMEGALOVIRUS (HHV-5) INFECTIONS
(Left) Reactive and inflamed
small bowel epithelium
with hemorrhage contains
an enlarged cell with
rominent granules .
Immunohistochemistry
was positive for CMV.
(Right) Reactive and mildly
inflamed colonic epithelium
is shown with scattered
cells suspicious for viral
cytopathic effect , which
stained positive for CMV by
immunohistochemistry.
(Left) Colonic epithelium
with reactive changes,
degeneration of epithelial
cells, and clear CMV viral
cytopathic effect are
shown. The scattered
epithelial cells with pale pink
cytoplasm are also infected
with CMV. (Right) Severe
villitis in a fetal demise shows
diffuse viral cytopathic effect
with CMV and severe
inflammation with villus
destruction .
(Left) Severe CMV
neumonia demonstrates
redominantly nuclear
staining for CMV antigen
by immunohistochemistry.
(Right) Nuclear and
cytoplasmic staining of
CMV-infected cells is shown
in an immunocompromised
atient with adjacent
infected cells positive for
antigen, which were not
histologically suspicious
cells.
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