Enterovirus D68

Enterovirus D68 (EV-D68), also known as enterovirus 68 (EV-68 or EV68), is a non-poliovirus, nonenveloped, positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV-D68 is transmitted person to person by contact with respiratory and gastrointestinal secretions.

Electron micrograph of a thin section of enterovirus D68 (EV-D68), showing the numerous, spherical viral particles. Courtesy of the CDC.

Electron micrograph of a thin section of numerous spherical enterovirus D68 (EV-D68) viral particles. Some of the particles appear to be "empty," missing their contents of single-stranded RNA. Courtesy of the CDC.

Whereas most of the approximately 100 species of enteroviruses primarily infect the gastrointestinal (GI) tract, EV-D68 has tropism for the respiratory tract. In vitro studies have shown that EV-D68 can affect a variety of cell lines, including neural tissue.[1]  It causes acute respiratory disease ranging from mild upper respiratory tract symptoms to severe pneumonia and has been associated with central nervous system infections with polioviruslike manifestations leading to an acute flaccid myelitis. 

In the outpatient setting, EV-D68 disease has manifested most commonly among persons younger than 20 years and adults aged 50-59 years. Inpatients have predominately been children.[2, 3]

EV-D68 was first isolated in 4 pediatric patients with lower respiratory tract infection in California in 1962.[4]

EV-D68, as with other enterovirus species, circulates seasonally in the summer and especially in fall months.[5, 6]

In August 2014, EV-D68 emerged as a cause of severe respiratory infections. Hospitals in Illinois and Missouri reported an increased incidence of rhinovirus and enterovirus infection, with 30 of 36 isolates from the nasopharyngeal secretions of patients with severe respiratory illness identified as EV-D68. Following these reports, an unusually high number of patients with severe respiratory illness were admitted to these facilities, presumably with EV-D68 infection.[3]

EV-D68 outbreaks have also been reported in Europe, Argentina, China, and Japan.[7, 8, 9, 10, 11, 12, 13]

Susceptible patients generally have underlying asthma and wheezing.

Enteroviruses typically spread through the fecal-oral route, although some serotypes, like EV-D68, also spread via respiratory secretions. The enteroviruses invade the submucosal tissues of the distal pharynx and alimentary tract. The incubation period varies from 3-10 days, leading to a transient minor viremia that delivers virus to lymphoid tissues. Ongoing viral replication results in major viremia that spreads the virus to targets such as the CNS.

For infection to take hold, the virus must recognize and attach to host cells. Sialic acid was proposed as a receptor for EV-D68 as some strains have been shown to have sialic acid dependent cell entry; however, it was seen that EV-D68 could also infect cells that lack sialic acid receptors suggesting that they can use an alternative receptor.[14, 15]

Once attached and endocytosed into the host cell, EV-D68 manipulates the host cell cycle in order to promote viral replication. The nonstructural viral proteins 3C and 3D appear to have a role in arresting the host cells in the G0/G1 phase, which is most favorable for EV-D68 replication.[16]

Enterovirus neurovirulence is believed to result from direct cellular destruction.[17, 18] In vitro neuron-specific intercellular adhesion molecule 5 (ICAM-5/telencephalin) was noted to be another cellular receptor for EV-D68.[19] EV-D68 pathogenesis was examined by Schieble et al[20] in suckling mice by intrathecal and intraperitoneal inoculation with 4 strains of EV-D68. CNS pathogenicity was observed only in mice inoculated with the Rhyne strain, showing limb tremor, weakness, paralysis, and death. This experiment is consistent with case reports of fatal meningoencephalitis resulting from EV-D68, serving as evidence of the neurovirulence of the virus. Ferrets with EV-D68 virus infection have been shown to have minimal clinical symptoms and have been proposed to be used as animal models for EV-D68 infection and pathogenesis.[21]

Immunology

The immune response to enteroviruses is mainly humoral, mediated by secretory immunoglobulin A (IgA) in nasal and alimentary secretions. Serum neutralizing immunoglobulin G (IgG) is detected 7-10 days after infection and persists for life after natural enteroviral infection. Macrophages also play an important role in viral clearance.[17, 18]

EV-D68 has been shown to avoid the innate immune system's responses via 3Cpro protein, which targets key players in both Toll-like receptor–mediated signaling and retinoic acid-inducible gene I-like receptor pathway.[22, 23]

Human EV-D68 is a small nonenveloped virus with single-stranded positive-sense RNA genome of 7.5 kilobases. It belongs to the enterovirus D species, within the Enterovirus genus in the Picornaviridae family.[4, 24] EV-D68 is acid-labile and behaves more like human rhinoviruses in its affinity for the respiratory tract.

Enterovirus D68 was first identified in California in 1962 in 4 pediatric patients with bronchiolitis and pneumonia.[4] Enterovirus surveillance in the United States between 1970 and 2005 identified only 26 cases of EV-D68 infection. In autumn 2009, in the context of pursuing respiratory virus surveillance during the H1N1 pandemic, a cluster of EV-D68 cases were detected in New York.[25]

A similar increase in detection of EV-D68 has been reported worldwide, although the increased incidence was believed to have resulted from the use of more highly sensitive virus identification methods such as polymerase chain reaction (PCR). Verifying stored respiratory samples from Yamagata, Japan and the Netherlands revealed EV-D68–positive samples in recent years.[26] In 2010, 40 cases were reported in Yamagata and 24 in the Netherlands.

The frequency of outbreaks may vary among countries.[26] In the Philippines, for example, EV-D68 outbreaks in the last decade appear to be cyclic in 2-year intervals: 2008-2009, 2011, and 2013-2014.[26] This may be partly due to differences in regional climate. In South Africa, 8 cases were detected between May 2000 and May 2001 in children aged 5-23 months. Three cases were reported from February to March 2010 in Senegal and 5 cases in June 2008 in Gambia. The climate in these countries consists primarily of dry and rainy seasons, but a much larger sample will be needed to assess the seasonal circulating patterns in non-temperate regions.[25]

In the United States, a large outbreak of EV-D68 occurred in 2014 with the CDC reporting 1395 people contracting the virus.[27] During this outbreak, pediatric hospitals identified EV-D68 as a possible cause for acute neurological illnesses and acute respiratory dysfunction.[28, 29]  A national surveillance of acute flaccid myelitis noted a biennial occurence with EV-D68 infection being associated during the peak years of incidence (2014, 2016, and 2018).[30, 31, 32, 33, 34]  Seven co-circulating strains of EV-D68 have been identified in the outbreak in 2014 by extracting genome sequences generated from RNA from nasopharyngeal swab supernatant or from virus isolates obtained by inoculation of nasopharyngeal supernatants into human rhabdomyosarcoma cells. The VP1 gene sequences of the 7 EV-D68 strains are most closely related to EV-D68 viruses detected previously in the United States, Europe, and Asia in recent years.[4]

In September 2014, a case of acute flaccid paralysis following enterovirus D68–associated pneumonia was reported in France.[24]

A few cases of human enterovirus as a cause of respiratory disease in Latin America have been reported. A study was conducted in 8 countries throughout Central and South America, where 3,375 nasopharyngeal swabs from subjects aged 1 month to 25 years (median age, 3 years) with influenzalike symptoms were collected; 84 samples (3%) were positive for human enterovirus via RT-PCR, of which EV-D68 was among the isolated serotypes.[35]

Between the 1960s and mid-1990s, EV-D68 underwent some genetic rearrangements. Such resulting phenotypic changes are thought to have a role in immune response modulation to the virus, which may aid in viral persistence.[25] Prior studies indicated that different phylogenetic lineages of EV-D68 co-circulate because of increased variability of the VP1 genomic region, leading to reduced cross-neutralizing antibodies raised against viruses of the major groups.[2]

EV-D68 circulates between summer and fall and appears to spread by close contact with infected people (cough, saliva, mucus, fomites).

Susceptible populations

Pediatric patients with a history of asthma, wheezing, or other underlying respiratory disease were shown to be at risk for more severe disease.[3] Children, especially those aged 1 month to 16 years, appear to be the most susceptible population based on most reports. They represented more than 80% of cases in both the Missouri and Illinois outbreaks (mean age, 4-5 years, respectively).[3] A 2014 report from the Netherlands found 95% of cases were in children.[25] Neurological disease has been described most commonly in males.[36]

The prognosis of EV-D68 infection varies, ranging from mild, self-limited respiratory infection to severe respiratory disease, especially in individuals with asthma.

In patients with CNS involvement, which is rare, residual neurologic deficits can persist. From a series of 23 cases of acute flaccid paralysis reported in California between 2012 and 2014, only 2 tested positive for EV-D68. The remaining cases had no clear etiology. Most cases require extended hospital stays (median, 17 days). Thirteen of 23 patients with available data had prolonged paralysis persisting at 60 days follow-up. In contrast, most patients who presented with mental status changes returned to baseline.[36]

Preventive measures are as follows[29] :

• Stay home if ill

• Wash hands with soap and water

• Avoid close contact with those who are ill

• Clean and disinfect frequently touched surfaces

The spectrum of clinical presentation of enterovirus D68 (EV-D68) infection can range from mild respiratory infection to severe disease, warranting intensive care unit admission, bi-level positive airway ventilation, or even intubation. Some cases have resulted in death.[3, 25]

Potential manifestations of EV-D68 infection are as follows:

• Acute onset

• Cough

• Fever (although a considerable percentage of cases without fever reported in the latest outbreaks)

• Rhinorrhea

• Sore throat

• Fatigue

• Headache

• Myalgia

• Dyspnea

• Diarrhea

• Bronchiolitis[2]

Physical examination may reveal the following:

• Wheezing

• Rhinorrhea

• Fever

• Hypoxemia[2]

Cases involving acute neurological illness may have the following characteristics[29, 36] :

• Preceding febrile illness with upper respiratory tract symptoms or GI prodrome occurring 3-16 days before onset of neurological illness

• Limb paresis or flaccid paralysis

• Ptosis, facial droop, dysarthria, dysphagia, diplopia

• Absence of sensory deficits

• Meningismus in cases of meningoencephalitis (one case described in 2008 in New Hampshire[26] )

In the evaluation of respiratory disease, obtain nasopharyngeal or oropharyngeal swabs and other respiratory specimens such as nasal wash or aspirates, which are high yield for diagnosis.

Initially, the assay used to diagnose enterovirus D68 was real-time RT-PCR (rRT-PCR) developed by the CDC Picornavirus Laboratory. This technique involves sequencing a region of the virus’s genome, which results in complicated large-scale screening.[38] However, in July 2015, a new assay was developed by researchers at the Washington University School of Medicine in St Louis. This assay is a rRT-PCR that is more sensitive than commercially available assays for enterovirus and rhinovirus detection. This new assay is also more specific for EV-D68, unlike the commercially available assays, which do not distinguish between the two. This newly developed assay was also able to detect divergent strains of EV-D68.[39]

Chest radiography should also be performed in patients with respiratory symptoms.

If neurological manifestations of paralytic disease or meningoencephalitis are evident, a lumbar puncture and cerebrospinal fluid (CSF) analysis are warranted.[24, 37] If flaccid paralysis is present, MRI of the spine can provide diagnostic data.[37] If cranial nerve manifestations are present, MRI of the brain should also be obtained.[37]

Chest radiographs show perihilar infiltrates, often with atelectasis when respiratory disease present.[3]

In cases of acute flaccid paralysis and cranial nerve dysfunction in children, MRI of the spine demonstrates nonenhancing lesions of the gray matter spanning multiple levels, and MRI of the brain has shown nonenhancing brainstem lesions most commonly localized to the dorsal pons. MRI with gadolinium can show enhancing of ventral nerve roots of the cauda equina.[29]

Cerebrospinal fluid typically shows a lymphocytic pleocytosis, normal glucose levels, and normal or mildly elevated protein levels, consistent with aseptic meningitis. RT-PCR of CSF yielded negative findings in one case series.[29]

Nasopharyngeal specimens testing with rRT-PCR is frequently the diagnostic tool used to identify EV-D68.

Blood cultures are negative for bacteria.

In outpatient settings, ensure that the patient has an asthma action plan to control asthma long-term and understands how to manage worsening asthma or attacks. Provide instructions concerning when to call his or her primary physician or to seek emergency treatment.[38]

Patients with severe respiratory infection should be hospitalized for supportive treatment, as follows:

• Oxygen if the patient is hypoxic
• Bronchodilator therapy if the patient presents with clinical evidence of bronchospasm
• Endotracheal intubation and mechanical ventilation may be needed in patients with impending respiratory failure

No specific intervention has been shown definitive efficacy in EV-D68 infections with acute flaccid myelitis. Human intravenous immunoglobulin (IVIG) may offer some benefit, as reported by Zhang et al, who found high levels of neutralizing antibodies to EV-D68.[40] Studies performed in mice with EV-D68 infection with paralytic myelitis showed that human IVIG reduced paralysis and decreased spinal cord paralysis, while fluoxetine had no effect and dexamethasone resulted in worse motor impairment and increased mortality and increased viral loads.[41] However, in a small review of 5 cases of poliolike syndrome, steroids, intravenous immunoglobulin, and plasma exchange showed no apparent clinical benefit.[42]

No data on the efficacy of antivirals in humans exist. Promising data have shown that rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, and novel diketopiperazines have in vitro activity against EV-D68.[43] Other antiviral drugs have been proposed as drug candidates for EV-D68 targetting different viral particles.[44, 45, 46]

Consider consultation with an infectious disease specialist and/or neurologist.

No vaccinations are available for EV-D68 infection. As no suitable animal model still exists for EV-D68, a full understanding of the pathogenesis and immune response against EV-D68 is incomplete. Studies are underway to establish animal models and the effect of EV-D68 on their immune system to investigate antiviral therapies and vaccines.[21, 47]

Plecoranil, pocapavir, and vapendavir are antiviral drugs with significant activity against a wide range of rhinoviruses and enteroviruses; however, these drugs have been tested with currently circulating strains of EV-D68 by the CDC, and none of them has activity against EV-D68 at clinically relevant concentrations.[38]

In vitro studies have demonstrated some promising results for developing antiviral medications, but in vivo studies are lacking.[44, 45] ref46}[48, 49]