- AFM Is a Polio-Like Disease Confirmed in 24 States Across the U.S.
- An Enterovirus Is Associated with AFM
- AFM Has No Cure, but a Preventative Vaccine Appears Possible
About a month ago, a tweet about an AFM outbreak in children in Pittsburgh caught my attention. Because I had never heard of AFM, I searched scientific literature, learning that the disease is somewhat similar to polio, and has sporadically occurred in clusters around the world for quite a few years. One of the causative agents of AFM seems to be an enterovirus that mutates into various genotypes and phenotypes, based on information gathered from genome sequencing, PCR-based analyses, and clinical data. Now, combatting AFM and avoiding potentially major outbreaks are issues of growing concern.
This blog on AFM is my attempt—as a non-expert in virology—to distill complicated science and epidemiology into commentary that fits into what’s trending in nucleic acids research. Having said that, I’ll now start with a brief synopsis of AFM as a disease and its presumptive causative agent, “enterovirus D68” (EV-D68), and I’ll finish with some information on a possible protective vaccine against this debilitating illness.
Basic Facts About AFM
In general, my go-to sources for authoritative, up-to-date information on diseases are the National Institutes of Health (NIH) website, and the Centers for Disease Control (CDC) and Prevention website. Each web page is thoroughly referenced, with convenient links to primary sources. Here is a brief synopsis of information that I found on AFM by searching “Acute Flaccid Myelitis” on the NIH and CDC sites.
Symptoms: AFM is a rare disease that affects the spinal cord, specifically the area of the spinal cord called gray matter. This causes the muscles and reflexes in the body to become weak. Symptoms of AFM include sudden (acute) weakness in the arm(s) or leg(s), along with loss of muscle tone (flaccid), and decreased or absent reflexes. In some cases, AFM can cause facial weakness, drooping of the eyelids, and difficulty swallowing, speaking, or moving the eyes. The most severe symptom of AFM is respiratory failure, which can happen when the muscles involved with breathing become weak. This can require urgent support and utilization of an assisted-breathing device, like the one pictured above.
Interested readers can access more information by perusing recent videos on AFM via YouTube. Most of these videos discuss a spike or outbreak of AFM in a particular state, and refer to AFM as a “polio-like” illness, an oversimplification that is widely used in mainstream media reports. Be forewarned that several posts among these AFM videos offer comments that I would politely describe as unscientific, if not plain bizarre.
Diagnosis: Most cases of AFM occur in children. Unfortunately for patients and parents, AFM can be difficult to diagnose because the symptoms are similar to other neurological diseases, such as Guillain-Barre syndrome (GBS), acute disseminated encephalomyelitis (ADEM), and transverse myelitis. Diagnosis may include an MRI of the spine, testing of the cerebral spinal fluid (CSF), tests checking nerve speed (nerve conduction velocity; NCV), and muscle response to nerve messages (electromyography; EMG). According to the CDC, as of October 31st 2018, there are 191 reported cases under investigation in 24 states across the U.S., of which 72 have been confirmed to be AFM cases.
Treatment: There is no specific treatment for AFM, but a neurologist specialized in treating brain and spinal cord illnesses may recommend certain interventions on a case-by-case basis. For example, neurologists may recommend physical therapy to help with arm or leg weakness caused by AFM. However, the extent of recovery varies – although some patients may make a full recovery, most have continued muscle weakness. The long-term outcomes of people with AFM remain unknown.
Causes: I was quite surprised at the CDC website’s statement that “AFM or similar neurologic conditions may have a variety of possible causes such as viruses, environmental toxins, and genetic disorders. Oftentimes, despite extensive lab tests, the cause of a patient’s AFM is not identified.” Given the sophistication of modern medicine and molecular diagnostics, I was expecting more definitive information. This in turn prompted me to research the reported virus-associated causes of AMF, which you can read about below.
EV-68 Is a Causative Agent for AMF
Enteroviruses are a genus of positive-sense single-stranded RNA viruses, and they are named based on their transmission-route through the intestine (enteric means intestinal). A simplified depiction of the basic structural elements of an enterovirus is shown here. Classification of viruses can be inherently complicated, and it is not a topic that can be easily discussed due to the evolution of naming conventions, which sometimes appear to be a seemingly incomprehensible mix of Latin or Greek nomenclature, letters, and numbers.
Human enteroviruses are currently classified into 12 species: enteroviruses A through J (excluding letter I), and rhinoviruses A through C. Enteroviruses isolated relatively recently are named with a system of consecutive numbers. For example, EV-D68 belongs to enterovirus D. EV-D68 has a genome that contains a single open-reading frame, coding for a poly-protein (P1), the precursor of four viral capsid proteins, VP1, VP2, VP3, and VP4, and seven non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. VP1 and VP3 are the major antigenic epitopes.
EV-D68 was first isolated in 1962 in California, from children with pneumonia and bronchiolitis. According to a June 2018 publication by Sun et al., there have been only 26 cases of documented EV-D68 respiratory disease in the U.`S. from 1970 to 2005. However, the upsurge of EV-D68 cases in the past few years showed clusters of infections in Europe, the Americas, Asia, Oceania, and Africa. In particular, more than 1,000 cases, including 14 deaths, were reported during an epidemic of EV-D68 infection in 2014 in the U. S., resulting in strong public attention toward this virus, as well as intensified research efforts on how to combat it.
A year later in 2015, Huang et al. reported carrying out a metagenomic shotgun sequencing protocol on clinical samples and negative controls, the results of which allowed for the assembly of 20 EV-D68 genomes: 6 complete, and 14 near-complete. A comparative genomic analysis revealed that EV-D68 strains circulating in the 2014 outbreak were significantly different from prior ones, and that they actually belong to a new clade. Importantly, two functional mutations in EV-D68 may alter its protease cleavage efficiency, thus leading to increased rate of viral replication and transmission.
In 2015, Zhuge et al. reported on the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (RT-PCR) developed by the CDC. Nasopharyngeal swab specimens from patients testing positive for rhinovirus or enterovirus were assessed using this EV-D68 RT-PCR, and the data were compared to results from partial sequencing analysis of the EV genome. The EV-D68 RT-PCR data showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. It was concluded that EV-D68 RT-PCR is a reliable assay for detection of EV-D68 in clinical samples, and it has the “potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.”
EV-D68 Is Spread in Multiple Ways
Although a number of whole-genome sequencing studies on EV-68 have now been reported in the context of comparative genomics, my inner “alarm bell” was triggered by a Lednicky et al. report. The researchers collected EV-D68 from classroom air using a filter-based air sampling method, and from environmental surfaces by swab sampling. Then, they amplified and sequenced the complete genome of the enterovirus. Here is a brief description of what they found and concluded:
EV-D68 was detected in 4-of-6 air sampler filters, and in 12-of-16 desk tops in a university classroom. cDNA synthesis was performed with avian myeloblastosis virus (AMV) reverse transcriptase and random hexamers on the viral nucleic acids extracted from filters or swabs, and PCR was performed using a panel of respiratory virus primers. Quantitative RT-PCR tests executed after the virus was identified indicated 400 to 5,000 genomic equivalents of EV-D68/m3 in the air samples. Viral RNA from the air sample with the highest concentration of virus was used for sequencing.
The researchers concluded that, “[a]s with our findings, high levels of airborne enteroviruses [have previously been] detected in a pediatric clinic, and this may be a common finding in indoor settings with enterovirus-infected individuals. Our work also suggests that young adults can produce airborne EV-D68 and raises the question of whether airborne transmission is important for spreading the virus.”
According to an article that I found in a journal on hygiene, EV-D68 can be found in bodily fluids such as saliva, mucous, sputum, and feces. It is transmitted through direct contact, including shaking hands, touching contaminated objects or surfaces, changing diapers of an infected person, or drinking water containing the virus. Following infection, the virus can be shed in stool for several weeks, and in the respiratory tract for up to 3 weeks. Shedding of EV-D68 can occur even in the absence of symptoms.
EV-D68 Vaccine Status
The Benjamin Franklin axiom that “an ounce of prevention is worth a pound of cure” is as true today as it was when Franklin said it, especially when referring to modern vaccines. I researched the status of vaccines to protect against AMF, and found the following report by Dai et al.
These researchers describe the development of a virus-like particle (VLP)-based experimental EV-D68 vaccine. They found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1, and that they were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro.
Importantly, passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. According to Dai et al., “these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.”
Along similar lines, Patel et al. have found that cotton rats are permissive to EV-D68 infection without virus adaptation. Three different strains of EV-D68 studied all showed the ability to produce neutralizing antibody upon intranasal infection or intramuscular immunization. Patel et al. concluded that “our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.”
An EV-D68 mRNA Vaccine Seems Feasible
In 2017, a high-visibility Nature publication by Pardi et al. demonstrated that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP), encoding the pre-membrane and envelope glycoproteins of Zika Virus (ZIKV), elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination.
In my opinion, this landmark proof-of-concept study, which I’m pleased to say used modified mRNA comprised of 1-methylpseudouridine-5′-triphosphate obtained from TriLink, should be readily extended to EV-D68. The work of Dai et al. mentioned above, together with the information gained from genomic sequencing of EV-D68, provide the conceptual basis for synthesis of modified mRNA encoding one or more antigenic proteins as potential vaccines. These candidates could then be tested in one or both of the animal models mentioned above.
Unfortunately, drug and vaccine companies tend to be reluctant to invest in research targeting rare diseases. Development of a vaccine against AFM requires adequate financial resources. One can only hope that such resources become available, perhaps through a charitable foundation, the NIH, or maybe even crowdfunding, artistically depicted here.
The NIH has taken the lead on placebo-controlled clinical trials for a ZIKV vaccine; however, as detailed in a September 14, 2018 Science news article, a steep drop in Zika cases has undermined this and other planned related trials. This has led researchers to consider trials in which subjects are deliberately exposed to ZIKV, but studies like these would be faced with serious ethical and safety issues. Given the rarity of AFM cases, clinical evaluation of the efficacy of a candidate vaccine against AFM would encounter similar issues.
As usual, your comments are welcomed.
After writing this blog, I became motivated to do a literature search for treatment of AFM. I found a 2017 publication by Tyler and coworkers titled Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis. This study evaluated 3 widely used empirical therapies for their ability to reduce the severity of paralysis in a mouse model of EV-D68 infection: (1) human intravenous immunoglobulin (hIVIG), (2) fluoxetine, and (3) dexamethasone.
Importantly, hIVIG, which was shown to contain neutralizing antibodies for EV-D68, reduced paralysis in infected mice and decreased spinal cord viral loads. Fluoxetine had no effect on motor impairment or viral loads. Dexamethasone treatment worsened motor impairment, increased mortality, and increased viral loads.
The following concluding statement from this 2017 publication is especially promising: “Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans.” In my opinion, of the 3 therapies investigated in this mouse model, treating patients with readily available hIVG seems to be the strongest course of action.