Case Reports

A Fatal Central Nervous System Enterovirus 68 Infection

Justin D. Kreuter, MD; Arti Barnes, MD; James E. McCarthy, MD; Joseph D. Schwartzman, MD; M. Steven Oberste, PhD;
C. Harker Rhodes, PhD, MD; John F. Modlin, MD; Peter F. Wright, MD

The anticipated eradication of poliovirus emphasizes the

Nneed
to identify other enteroviral causes of severe central
nervous system disease. Enterovirus 68 has been implicated
only in cases of respiratory illness. We therefore report a
case of fatal meningomyeloencephalitis caused by enterovirus 68 in a 5-year-old boy, which required neuropathology, microbiology, and molecular techniques to diagnose.
(Arch Pathol Lab Med. 2011;135:793796)

REPORT OF A CASE
The patient was a previously healthy 5-year-old boy with upto-date immunizations who initially sought care for headache,
low-grade fever, sore throat, and unilateral neck tenderness in
the fall of 2008. He had no tick exposure or travel outside of New
Hampshire. A number of classmates reportedly had coldlike
illnesses. During the following 2 days, he experienced myalgia
and progressive weakness of his arms (right arm to a greater
extent than left) and a change in the timbre of his voice. Four days
after onset he visited his primary care provider who believed he
had a nonspecific viral illness. He had a white blood cell count of
10 900/mL (80% neutrophils, 5% bands, and 12% lymphocytes).
Serum electrolyte and liver function test results were normal. The
erythrocyte sedimentation rate was 16 mm/h. In the next few
hours the patient developed bowel and bladder incontinence and
the inability to walk. Later that evening he was found in bed,
apneic and unresponsive, with intermittent tonic posturing of the
upper extremities. He had a cardiac arrest en route to the
hospital, and on admission to the pediatric intensive care unit he
remained unresponsive and had a temperature of 32.5uC, an
arterial blood pH of 6.5, and a PCO2 of 102 mm Hg. A computed
tomography scan of the brain showed diffuse cerebral edema
with tentorial herniation. A chest x-ray showed a hazy opacity of
the right lower lobe. As clinical criteria for brain death were met,
life support was withdrawn and a full autopsy performed.

POSTMORTEM LABORATORY FINDINGS

Cerebrospinal fluid (CSF) obtained by cisternal puncture showed slight xanthochromia; a nucleated cell count
of 2094/mL (54% lymphocytes); glucose, 2 mg/dL; and
protein, 368 mg/dL. Tissue and CSF were sent for
Accepted for publication August 16, 2010.
From the Departments of Pathology (Drs Kreuter, Schwartzman, and
Rhodes), Medicine (Dr Barnes), and Pediatrics (Drs McCarthy, Modlin,
and Wright), Dartmouth-Hitchcock Medical Center, Lebanon, New
Hampshire; and the Department of Virology, Centers for Disease
Control and Prevention, Atlanta, Georgia (Dr Oberste).
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Justin D. Kreuter, MD, Department of Pathology, Dartmouth-Hitchcock Medical Center, 1 Medical Center Dr, Lebanon, NH
03756 (e-mail: justin.d.kreuter@hitchcock.org).
Arch Pathol Lab MedVol 135, June 2011

diagnostic studies at the Dartmouth-Hitchcock Medical

Center (Lebanon, New Hampshire), the New Hampshire
Public Health Laboratory (Concord, New Hampshire),
and the Centers for Disease Control and Prevention
(CDC), Polio and Picornavirus Branch (Atlanta, Georgia).
Findings from the bacterial cultures of the frontal cortex,
CSF, lung, stool, and urine were all negative. Results of
viral cultures (herpes simplex virus, cytomegalovirus,
varicella zoster virus, and enterovirus) of the frontal lobe,
lung, and CSF were negative. Screening of CSF for West
Nile and Eastern equine encephalitis viruses by reverse
transcriptasepolymerase chain reaction (RT-PCR) yielded negative results at the state laboratory. The CDC
identified enterovirus 68 in the patients CSF but not the
frontal lobe, using VP1-specific RT-PCR and sequencing.1
An attempt to amplify enterovirus 68 from formalin-fixed
lung tissue was unsuccessful owing to RNA degradation.
Immunoglobulin (Ig) levels (IgG, 505 mg/dL; IgA, 44 mg/
dL; and IgM, 62 mg/dL) were all normal for the patient9s
age.
GENERAL AUTOPSY FINDINGS
The patients lungs were 3 times the normal weight and
diffusely firm. Microscopic sections showed acute mixed
pneumonia with hemorrhage (Figure, A). The spleen was
twice the normal weight and florid follicular hyperplasia
was observed microscopically. Sections of the thymus and
bone marrow showed normal immune architecture.
NEUROPATHOLOGIC FINDINGS
Postmortem gross examination of the brain demonstrated profound edema and normal-appearing leptomeninges. The brainstem was grossly unremarkable, although multiple red-brown discolorations averaging
7 mm in greatest diameter were identified in the dentate
nucleus bilaterally. Microscopic examination of the
hippocampus revealed hypereosinophilic neurons with
pyknotic nuclei, consistent with hypoxic-ischemic injury.
Microscopy of the meninges, cerebellum, midbrain, pons,
medulla, and cervical cord demonstrated extensive
lymphocytic meningomyelitis and encephalitis, characterized by prominent neuronophagia in motor nuclei (Figure,
B).
IMMUNOHISTOCHEMICAL AND SPECIAL
STAIN FINDINGS
Immunochemical stains for CD3 in the spinal cord
highlighted a diffuse parenchymal infiltration of T lymphocytes in and around motor nuclei (Figure, C). The
CD20 stain in the spinal cord identified B lymphocytes
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793

A, Lung shows severe acute inflammation consistent with acute pneumonia. B, Anterior spinal cord shows diffuse parenchymal lymphocytic host
response with neuronophagia of motor neurons by infiltrating lymphocytes. C, Anterior spinal cord characterizes the parenchymal lymphocytes as T
cells. D, Anterior spinal cord demonstrates the predominantly perivascular location of B cells. E, Anterior spinal cord demonstrates the lack of
activation of the apoptotic pathway by motor neurons. F, Anterior spinal cord demonstrates the T-cell cytolytic protein located on the motor neurons
(hematoxylin-eosin, original magnifications 3100 [A] and 3200 [B]; anti-CD3 antibody, original magnification 3100 [C]; anti-CD20 antibody,
original magnification 3100 [D]; anticaspase-3 antibody, original magnification 340 [E]; anti-perforin antibody, original magnification 3200 [F]).

794

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Enterovirus 68 InfectionKreuter et al

predominantly localized to the perivascular areas (Figure,

D). Antibody staining for caspase-3, a component of
apoptotic cell death, was negative in anterior spinal cord
a-motor neurons (Figure, E). Most a-motor neurons were
positive for perforin, the cytolytic protein used by CD8 T
cells and natural killer cells to cause cell death (Figure, F).
Gomori methenamine silver and Gram stain results were
negative in lung sections. Immunohistochemical studies
of lung and spinal cord with an available, but unvalidated,
antienterovirus 68 horse polyclonal antiserum were not
definitive.
COMMENT
This child presented with acute neurologic impairment,
flaccid paralysis, and pneumonia. The patients lumbar
puncture was consistent with aseptic meningitis when
correcting for postmortem decrease in glucose.2 Pertinent
negative findings include negative findings for bacterial
cultures, viral cultures, special stains for fungi and
bacteria and negative PCR results for 2 recognized causes
of encephalitis in New Hampshire (West Nile and Eastern
equine encephalitis viruses). Identification and sequencing of enterovirus 68 in the spinal fluid, combined with the
characteristic histopathologic pattern of enteroviral central nervous system infection, established this virus as the
cause of the fatal illness.3
The identification of the pathogen as enterovirus 68 is
based on the nucleotide sequence of the viral DNA present
in the patient, specifically the portion of the viral genome
region encoding the VP1 capsid protein. The sequence in
this region has been shown to correlate with enterovirus
serotype, as determined by antigenic methods.4 In this
case, a portion of VP1 (,350 nucleotides) was amplified
by seminested RT-PCR and the sequence was compared
with sequences in a database representing all known
enterovirus serotypes, using a previously described
method.1 The VP1 sequence was unique (not identical to
that of any other strain in the laboratory) and it was 85.0%
identical to the EV68 prototype strain, indicating a
successful identification (strains of the same type share
$75% nucleotide identity in this region).
The human enteroviruses are classified into 4 species in
the genus Enterovirus on the basis of their genetic
relationships.5 Human enterovirus A (HEV-A) and HEVC each contain some of the coxsackie A viruses and
several of the more recently numbered enteroviruses, and
polioviruses are classified within HEV-C. The coxsackie B
viruses, echoviruses, and most of the numbered enteroviruses are in HEV-B, while HEV-D includes EV68, EV70,
and EV94. Enterovirus 68 has not been previously
reported as a cause of neurologic disease. Although
enteroviruses are a well-recognized cause of aseptic
meningitis,3 and can cause disseminated disease in the
newborn6 and focal encephalitis,7 the identification of a
new enterovirus agent as a cause of serious neurologic
disease is noteworthy. The presenting symptoms of this
case, with asymmetric paralysis, and the histologic
findings of neuronophagia of motor nuclei in the anterior
horn cells are similar to disease caused by the 3 poliovirus
serotypes.
The pathologic findings in the brainstem and the
severity of disease are most analogous to viral meningitis,
acute motor neuron disease, and brainstem encephalitis
that complicate infections with enterovirus 71, a species A
enterovirus.8 In fact, the sudden cardiovascular collapse
Arch Pathol Lab MedVol 135, June 2011

observed in our patient with enterovirus 68 brainstem

encephalitis is strikingly similar to a rapidly fatal course
complicating some cases of acute enterovirus 71 encephalitisthat has been attributed to rapid onset of neurogenic pulmonary edema.911 In this case, the postmortem
lung findings demonstrated pneumonia rather than
pulmonary edema. Previously, enterovirus 68 has been
isolated primarily from the respiratory tract. It has been
described as having phenotypic characteristics of a
rhinovirus, as it is more acid-sensitive and thermolabile
than other enterovirus serotypes.12,13 Although we failed to
identify enterovirus 68 in the lungs of this patient, we used
a suboptimal cell mix for replication (R-Mix, Diagnostic
Hybrids, Athens, Ohio) and higher than optimal temperature for enterovirus 68 detection in tissue culture. The
radiologic and pathologic evidence of pneumonia without
a bacterial etiology suggests that systemic spread of
enterovirus 68 from a respiratory source may have been
a component of the pathogenesis of this case. Most efforts
at identification of causes of polio-like illnesses are based
on enteric shedding of viruses, such that enterovirus 68
associated illnesses, if primarily replicating in the respiratory tract, might be missed. The normal immunoglobulin levels and unremarkable medical history make it
unlikely that this patient had a B-cell defect that
predisposed him to more serious enterovirus disease.14
Alternative causes of viral encephalitis were ruled out by
molecular diagnostic techniques and culture and histopathologic findings.
The immunopathogenesis of enteroviral infection is not
completely understood. What has been learned comes
from the study of myocarditis.15 A competent immune
system with intact T-cell function is necessary for viral
clearance. In general, B cells inhibit viral replication and
geographically contain the virus with the help of
macrophages, whereas T cells enter into these areas of
viral infection, resulting in tissue damage and cell death.
In this case report, abundant T cells were seen infiltrating
motor nuclei of the brain, presumably leading to the
observed cell death of motor neurons (Figure, C). To
determine whether motor neurons were dying as a result
of apoptosis versus necrosis, staining for caspase-3 (a
proapoptotic protein) and perforin (a pore-forming
protein that causes necrosis) were performed. Motor
neurons showed no staining with anticaspase-3 but
stained strongly with anti-perforin, consistent with the
proposed cytotoxic role played by T cells in this patients
infection (Figure, E and F).
Enterovirus 68 appears to be a rare cause of neurologic
disease. The CDC Picornavirus Laboratory has identified
only 1 other enterovirus 68 from CSF in the past 10 years.
Nevertheless, identification of enterovirus 68 and other
novel enterovirus neuropathogens will increase our
awareness of their clinical role and pathogenesis and help
establish their true incidence.
We thank Jason Pettus, MD, for his thorough postmortem
examination and David Beck, HTL(ASCP) for his expertise in
immunohistochemical staining.
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