A UC San Francisco-led research team has detected the immunological remnants of a common seasonal virus in spinal fluid from dozens of patients diagnosed with acute flaccid myelitis (AFM) – a polio-like illness that causes permanent, sometimes life-threatening paralysis in young children.
The findings provide the clearest evidence to date that AFM is caused by an enterovirus (EV) that invades and impairs the central nervous system.
The study was published October 21, 2019 in Nature Medicine.
AFM, which begins with cold-like symptoms and progresses to limb weakness and paralysis in a matter of days, was first documented in 2012.
Since then, AFM outbreaks have occurred every other year, with more than 500 confirmed cases recorded so far.
But because scientists have had trouble pinpointing a cause, AFM has been the subject of contentious debate within the medical community.
Mounting evidence implicated EVs as the likely culprit – specifically the so-called D68 and A71 strains of the virus.
EV outbreaks are common and normally cause nothing more severe than cold-like symptoms or the rash-producing hand, foot and mouth disease.
Scientists started to notice, however, that EV outbreaks coincided with spikes in AFM.
They also found that respiratory samples from children diagnosed with AFM often tested positive for EVs.
Plus, laboratory studies found that these strains caused paralysis in mice.
But many experts remained skeptical of the enterovirus hypothesis, instead proposing that AFM is an autoimmune disorder or is caused by some other, as-yet-undiscovered virus.
These EV skeptics argued that that the evidence linking the virus to AFM was circumstantial, because the virus could not be found in 98 percent of AFM patients who had their spinal fluid tested.
They maintained that until there was ample evidence of the virus invading the human nervous system, the link between EVs and AFM remained unproven.
“People were hung up on the fact that enteroviruses were rarely detected in the cerebrospinal fluid of AFM patients.
They wanted to know how someone could get neurologic symptoms with no virus detectable in their central nervous system,” said Michael Wilson, MD, associate professor of neurology, member of the UCSF Weill Institute for Neurosciences, and senior author of the new study.
“If we could detect something specific to a virus in in the spinal fluid of AFM patients, we would feel more secure claiming that the neurologic symptoms of the disease are virally mediated.”
The group first searched for the virus directly in spinal fluid using advanced deep sequencing technologies, but this sort of direct detection of the virus failed, as it had previously.
Therefore, to find evidence of the missing virus, Wilson and his collaborators – researchers at the Chan Zuckerberg Biohub, the Centers for Disease Control and Prevention, the California Department of Public Health, the University of Colorado, Boston Children’s Hospital and the University of Ottawa – used an enhanced version of a virus-hunting tool called VirScan, first developed at Harvard Medical School in the laboratory of Stephen J. Elledge, Ph.D.
VirScan, which is a customized version of a Nobel Prize-winning technique called phage (rhymes with “beige”) display, allowed Wilson’s team to probe the spinal fluid of AFM patients for signs of an immune response against enterovirus and thousands of other viruses simultaneously.
“When there’s an infection in the spinal cord, antibody-making immune cells travel there and make more antibodies. We think finding antibodies against enterovirus in the spinal fluid of AFM patients means the virus really does go to the spinal cord. This helps us lay the blame on these viruses,” said Ryan Schubert, MD, a clinical fellow in UCSF’s Department of Neurology, a member of Wilson’s Lab, and lead author of the new study.
The researchers created molecular libraries consisting of nearly 500,000 small chunks of every protein found in the over 3,000 viruses known to infect vertebrates (including humans), as well as those that infect mosquitoes and ticks (an effort to rule out disease transmission through their bites).
They then exposed these molecular libraries to spinal fluid obtained from 42 children with AFM and, as a control, 58 who were diagnosed with other neurological diseases. Any chunks of viral protein cross-reacting with any antibodies present in the spinal fluid would provide evidence for a viral infection in the central nervous system.
Antibodies against enterovirus were found in the spinal fluid of nearly 70 percent of AFM patients; less than 7 percent of non-AFM patients tested positive for these antibodies. Furthermore, because spinal fluid from AFM patients did not contain antibodies against any other virus, every other known virus could be eliminated as a possible culprit. These results were confirmed using more conventional lab techniques.
“The strength of this study is not just what was found, but also what was not found,” said Joe DeRisi, Ph.D., professor of biochemistry and biophysics at UCSF, co-president of the Chan Zuckerberg Biohub, and co-author of the new study. “Enterovirus antibodies were the only ones enriched in AFM patients. No other viral family showed elevated antibody levels.”
Though the study provides the most robust evidence so far that enteroviruses cause AFM, many questions around AFM and these viruses remain unanswered.
For example, though the AFM-causing enterovirus strains – EV-D68 and EV-A71 – were identified decades ago, they only recently seemed to have gained the ability to cause paralysis, with the D68 strain in particular responsible for the most severe cases of AFM.
“Presumably there are changes that are causing the virus to be more neurovirulent, but no one knows for sure what they are,” Schubert said.
“Because the virus is found in such low amounts, if at all, it’s hard to zero in on the differences between an A71 virus that causes routine hand, foot, and mouth disease and one that causes AFM.”
Also, because enteroviruses are extremely common, scientists are still trying to figure out why fewer than 1 percent of infected children get AFM, and they’re also trying to understand why children are the only ones affected. “We don’t know for sure why children get paralysis and adults don’t,” Schubert said.
“The thinking is that young children have low immunity to the virus that increases as they get older, so we see the most severe effects in children around the age of two. But more work needs to be done to understand AFM.”
For study co-author Riley Bove, MD, answering these unresolved questions is a deeply personal mission. Bove, an assistant professor of neurology and member of the UCSF Weill Institute for Neurosciences, is the mother of a child who was diagnosed with AFM.
In the summer of 2014, Bove’s entire family came down with what seemed to be a severe cold. Everyone recovered except Bove’s then four-year-old son.
Just days after the onset of the cold-like symptoms, he started experiencing difficulty breathing. Soon, he was paralyzed from head to toe and had trouble breathing on his own.
Today, Bove’s son is a thriving nine-year-old, but she says the physical and emotional effects of AFM will be with him the rest of his life. “For every family with a child diagnosed with AFM, the long-term consequences of the disease remain the top issue,” she said.
Bove hopes that the new study will lead to a scientific consensus around enterovirus as the cause of AFM, since this a key step on the road to improved diagnostics and the development of a vaccine for the illness.
“Public health education is important, but it’s not enough to prevent AFM,” Bove said. “The virus is too common to avoid. A vaccine is the only way to meaningfully prevent the disease.”
For now, there’s no way to prevent or treat AFM. But if it follows the biennial pattern first established after the 2012 outbreak, AFM cases may spike again next year.
“We’re all holding our breath for 2020,” Schubert said.
Acute Flaccid Myelitis Associated with Enterovirus D68 in Children, Argentina, 2016
After a 2014 outbreak of severe respiratory illness caused by enterovirus D68 in the United States, sporadic cases of acute flaccid myelitis have been reported worldwide.
We describe a cluster of acute flaccid myelitis cases in Argentina in 2016, adding data to the evidence of association between enterovirus D68 and this polio-like illness.
We report a cluster of acute flaccid myelitis (AFM) cases in Buenos Aires, Argentina, in 2016. AFM was defined as acute flaccid paralysis (AFP) with magnetic resonance imaging (MRI) showing lesions predominantly affecting the gray matter of the spinal cord (1). We prospectively studied all patients with AFP who were admitted to Hospital de Niños “Ricardo Gutiérrez” in Buenos Aires during April 24–August 24, 2016, under the Argentine National Surveillance Acute Flaccid Paralysis Program for poliovirus as part of the World Health Organization AFP Program in the Americas. We obtained fecal samples or rectal swab specimens, serum samples, nasopharyngeal swab specimens, and cerebrospinal fluid (CSF) samples.
Fecal samples were tested at the National Reference Center for the Argentine National Surveillance Acute Flaccid Paralysis Program for enterovirus, including wild-type and vaccine-derived poliovirus. We screened clinical samples for enterovirus D68 (EV-D68) using a panrhinovirus and enterovirus nested PCR of enterovirus targeting the 5′ untranslated region (2). We purified the amplified products and prepared them for Sanger sequencing. We performed BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgiExternal Link) of GenBank sequences to identify which picornavirus was present. We obtained viral protein 1 partial sequences as previously described (3). In addition, we studied a wide panel of viruses (parainfluenza virus 1, 2, and 3; influenza A/B; respiratory syncytial virus; adenovirus; metapneumovirus; rhinovirus; varicella zoster virus; herpes simplex virus; cytomegalovirus) by reverse transcription PCR (RT-PCR) and studied bacteria by culture. We performed MRI and electromyography for all patients.
Fourteen children were admitted with AFP during April–August 2016. Six were confirmed to have AFM by case definition; the other 8 had alternative diagnoses, including Guillain-Barré syndrome (3), influenza virus myositis (2), encephalitis by echovirus (in 1 child with Down syndrome), acute transient hip synovitis (1), and transverse myelitis (1). Patients’ clinical, demographic, and outcome findings are shown in Table 1, diagnostic findings in Table 2.
In 4 (66.7%) of 6 patients, we confirmed EV-D68 infection by nested RT-PCR. In 1 patient, enterovirus was detected but not typed; in 1 patient, no agent was detected. All patients had distinctive neuroimaging changes. We followed confirmed AFM cases for 6 months to assess clinical improvement.
The median age of patients with AFM was 3.9 (range 1–5) years; 4 (66.7%) of the 6 were female, and 3 (50%) had a history of asthma. All patients had prodromal signs or symptoms before onset of neurologic symptoms: 100% had upper respiratory tract infection (URTI); 4 (66.7%) had fever; and 1 (16.7%) had vomiting and abdominal pain. Neurologic symptoms appeared 1–11 (median 2) days after URTI symptoms.
Results of hematology and chemistry analysis were normal for 5 (83%) patients. Patient 1 had leukocytosis (leukocytes 18,000 cells/mm3, with 82% neutrophils) and elevated levels of alanine aminotransferase (103 IU/L [reference 10–43 IU/L]), aspartate aminotransferase (97 IU/L [reference 10–35 IU/L]), and creatine kinase (6,591 IU/L [reference 24–170 IU/L]). During follow-up, patient 1 showed an increased creatine kinase level that could not be related to enterovirus infection.
All confirmed AFM case-patients showed T2 gray matter hyperintensity within the spinal cord on MRI. Electromyography showed early signs of denervation and low motor neuron function in all 5 patients in whom the test could be done. Specimen collection was performed 9.5 (range 3–30) days after URTI symptoms started and 7.5 (range 1–18) days after onset of neurologic symptoms.
We identified enterovirus using nested RT-PCR of nasopharyngeal samples in 5 (83%) of 6 patients; 4 (80%) of 5 were typed as EV-D68, but in 1 patient (20%) the viral load was too low for typing. We identified EV-D68 in 2 (33%) of 6 fecal specimens. We performed molecular characterization of EV-D68 strains based on phylogenetic analyses of a partial VP1 genomic region (Figure).
Results of nested RT-PCR for enterovirus were negative for all CSF samples; results of the respiratory virus panel were negative for all patients. Neither bacteria nor fungus were isolated in blood or CSF samples. Serum PCR to identify herpes simplex virus, varicella zoster virus, and cytomegalovirus also yielded negative results.
Intravenous immunoglobulin was empirically infused in 5 (83%) patients; 2 (33%) received systemic corticosteroids. Three patients required intensive care unit admission. All patients had neurologic sequelae: persisting palsy in >1 limbs and atrophy of muscles with a shortening of limbs. Two patients required chronic noninvasive ventilatory support during 6 months of follow-up. No patients died.
AFM has been associated with different etiologic agents (1). EV-D68 is a nonpolio enterovirus characterized by affinity for α2–6-linked sialic acids typically found in the upper respiratory tract, making the respiratory tract the preferred target for EV-D68 replication, unlike most enteroviruses, which replicate in the gut (1,4). Although there is no definitive evidence of causality between EV-D68 and AFM, since the 2014 EV-D68 respiratory outbreak in North America, AFM cases possibly associated with EV-D68 have been reported in the United States, Canada, Australia, Norway, Great Britain, and France (1,5). We report a cluster of AFM associated with EV-D68 in Argentina; another institution in Argentina (Hospital Garrahan) has also reported a case series of AFM (6,7).
The cluster in this report occurred over a 3-month period, during the 2016 autumn–winter season, which is the typical enterovirus season in Buenos Aires. Clinical and neurologic findings were similar to those of cases reported in other countries, including URTI preceding the neurologic features (4,8,9). Patients were admitted with asymmetric, acute, and progressive weakness of limbs; areflexia; and muscle pain. These symptoms have been reported as polio-like syndrome; however, testing and MRI should be performed for multiple viruses, including enteroviruses and EV-D68, to detect distinctive spinal cord lesions. No sensory sensitivity involvement was observed. Two patients had cranial nerve dysfunction. Laboratory findings were similar to those previously described, including CSF abnormalities (1,4,8).
Different hypotheses to explain difficulties in isolation of EV-D68 have been reported (4). It is possible that most of the nasopharyngeal specimens in previous studies and in our cluster were taken after 7 days of URTI, when the viral load is usually low, as reported by Imamura et al. (10). In our case series, enterovirus was identified in respiratory secretions in 5 (83.3%) of 6 patients, even though specimen collection was performed >7 days (mean 9 days) after AFM onset (in 1 patient, viral load was too low for genotyping). The negative nasopharyngeal specimen was collected at 18 days after onset.
Isolation of EV-D68 in fecal samples is uncommon because the virus is both heat and acid labile (1). However, in 2 (33.3%) of our 6 patients, EV-D68 was identified in fecal samples.
Reported rates of CSF detection of known neurotropic enteroviruses, such as polioviruses and enterovirus A71, are as low as 0%–5%, although viruses could be detected in brain or spinal cord tissue (4,11). A recent mouse model of AFM caused by EV-D68 showed that EV-D68 infects anterior horn motor neurons, resulting in motor neuron death (9). In our series, CSF samples tested negative for EV-D68 and other pathogens.
No specific treatment for EV-D68 AFM is available; the US Centers for Disease Control and Prevention recommends only support measures (7,12). Zhang et al. demonstrated that commercial immunoglobulin contained high levels of neutralizing antibodies against EV-D68 strains during the 2014 outbreak in the United States (13). No vaccines are available.
EV-D68 belonging to subclade B3 was identified in our cluster by molecular sequencing. This subclade was associated with EV-D68 circulation in the United States and Europe in 2016 (14).
We show a cluster of AFM associated with EV-D68 in Argentina. Our findings contribute to global evidence of EV-D68 as a possible cause of localized polio-like illness.
Dr. Carballo is a pediatric infectious diseases specialist at the Hospital de Niños “Ricardo Gutierrez” in Buenos Aires. Her research interests are pediatric infectious diseases.
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More information: Pan-viral serology implicates enteroviruses in acute flaccid myelitis, Nature Medicine (2019). DOI: 10.1038/s41591-019-0613-1 , https://nature.com/articles/s41591-019-0613-1
Journal information: Nature Medicine
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