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About viruses and viral disease

EV-D68

Enterovirus D68 and childhood paralysis

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EV-D68The Centers for Disease Control and Prevention thinks that viruses play a role in the childhood paralysis called acute flaccid myelitis (AFM). The finding of antibodies to enterovirus D68 (EV-D68) in the cerebrospinal fluid of patients with AFM strengthens the link between infection with this virus and AFM.

Acute flaccid myelitis (AFM), which mainly occurs in children, involves weakness of the arms and legs and may include other symptoms such as inability to breathe or swallow. There have been outbreaks of AFM in the US in 2014, 2016, and 2018, mainly in the late summer and early fall. The outbreaks of AFM are associated with infection with enteroviruses.

AFM was first defined in 2014 after reports of limb weakness in children across the US during an outbreak of respiratory disease caused by EV-D68 (pictured). AFM may also be associated with infections caused by other viruses, such as enterovirus A71, Coxsackievirus A16, West Nile virus and adenovirus.

Of the 233 patients with confirmed AFM in 2018, EV-D68 was the most frequently detected virus, mainly in respiratory samples. Confirming a viral etiological agent of AFM would be greatly strengthened by finding virus in the cerebrospinal fluid (CSF). However in 2018 only two samples of cerebrospinal fluid from AFM patients were found to be positive for viral RNA: one for EV-D68 and one for EV-A71. There may be multiple reasons for the failure to detect viral RNA in CSF, including clearance of virus by the time AFM is diagnosed, or very low levels of viral genomes.

An alternative to searching for viral genomes in CSF is to identify anti-viral antibodies, which may be produced during infection of the brain and spinal cord. Peptide microarrays were used to detect antibodies against enteroviruses in CSF of AFM patients. In this method, 160,000 unique peptides from the capsid proteins of all known human enteroviruses were spotted on slides. CSF samples were incubated with the slides and binding of antibodies to peptides was quantified. The results revealed antibodies to an 18 amino acid peptide of capsid protein VP1 in serum and CSF of 11/14 AFM patients and 3/26 controls. This amino acid sequence is known to be conserved among diverse human enteroviruses.

More importantly, antibodies to a 22 amino acid sequence in VP1 of EV-D68 were identified in CSF of 6/14 AFM, and in 8/11 serum samples. No immunoreactivity to this peptide was detected in samples from 26 controls.

Detection of antibodies in the CSF is not diagnostic of CNS infection because antibodies are known to pass from blood to CSF. The normal ratio of antibodies in blood to CSF is 250:1. Consequently, for diagnostic purposes, both serum and CSF should be analyzed for the presence of antibodies.

The authors of the study acknowledge this issue, noting that they do not have sufficient simultaneously collected serum and CSF samples to exclude that antibodies they detected in the CSF simply came from the blood. They note that of the 16 control patients with serum antibodies to enteroviruses, only 3 also had such antibodies in CSF. Furthermore, in a study of unexplained encephalitis in India, of 77 children with serum antibodies to enteroviruses, only 3 also had EV antibodies in CSF. For these reasons the authors do not believe that the anti-EV-D68 antibodies they have detected in CSF have come from the blood.

How can it be definitively proven that EV-D68 causes AFM? With an outbreak of AFM expected in the late summer of 2020, there should be ample opportunity to obtain numerous clinical samples – nasopharyngeal wash, serum and CSF – to examine for the presence of EV-D68 antibodies and viral genomes.

Enterovirus D68 infections in North America

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EV-D68
Enterovirus D68 by Jason Roberts

An outbreak of respiratory disease caused by enterovirus D68 began in August of this year with clusters of cases in Missouri and Illinois. Since then 691 infections have been confirmed in 46 states in the US.

The number of confirmed infections is likely to increase in the coming weeks, as CDC has developed a more rapid diagnostic test. Previously it was necessary to amplify the viral genome by polymerase chain reaction, followed by nucleotide sequencing to determine the identity of the agent. The new test utilizes real time, reverse transcription PCR which is specific for the EV-D68 strains that have been circulating this summer.

Since its discovery in California in 1962, EV-D68 has been rarely reported in the United States (there were 26 isolations from 1970-2005). Beginning in 2009 it was more frequently linked to respiratory disease outbreaks in North America, Europe, Asia, and Africa. It seems likely that the virus was always circulating, but we never specifically looked for it.

The current EV-D68 outbreak is the largest ever reported in North America. Enterovirus infections are not rare – there are millions every year in the US – but why EV-D68 has been so frequently isolated this year is unknown. One possibility is that the CDC, after the initial outbreak in August 2014, began looking specifically for the virus.

Sequence analysis of the EV-D68 viral genomes indicate that 3 different strains are involved in the US outbreak. These viruses are related to EV-D68 strains that have previously circulated in the US, Europe, and Asia. The sequences are available at GenBank as follows: US-IL-14/18952, US-KY-14/18951, US-MO-14/18950, US-MO-14/18949, US-MO-14/18948, US-MO-14/18947, and US-MO-14/18946.

Most of the illness caused by EV-D68 in the US has been respiratory disease, mainly in children. Five of the 691 confirmed EV-D68 cases were fatal, but whether the virus was responsible is not known.

There have also been some cases of polio-like illness in children in several states associated with EV-D68. In Colorado the virus was isolated from four of 10 children with partial paralysis and limb weakness. Previously there had been one report of an association of EV-D68 with central nervous system disease. In this case viral nucleic acids were detected in cerebrospinal fluid. EV-D68 probably does not replicate in the human intestinal tract because the virus is inactivated by low pH. If the virus does enter the central nervous system, it may do so after first replicating in the respiratory tract, and then entering the bloodstream.

There are no vaccines or antivirals to prevent or treat EV-D68 infection. Most infections will resolve without intervention save for assistance with breathing. As the fall ends in North America, so will infections with this seasonal virus.

Acute flaccid paralysis of unknown etiology in California

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EV-D68
Enterovirus D68 by Jason Roberts

In February 2014 I wrote about children in California who developed a poliomyelitis-like paralysis, also called acute flaccid paralysis or AFP. However, the cause of this paralysis was not known. The CDC has released its study of these cases and concludes “The etiology of AFP with anterior myelitis in the cases described in this report remains undetermined.”

A total of 23 cases of AFP* in California were reported to CDC during the period June 2012 through June 2014. These cases were from diverse geographic regions of the state. Specimens from 19 of the patients were available and tested for poliovirus, aroboviruses, herpesviruses, parechoviruses, adenoviruses, rabies virus, influenza virus, metapneumovirus, respiratory syncytial virus, parainfluenza viruses, Mycoplasma pneumoniae, Rickettsia, and amoebas. Rhinovirus was detected in one patient, and enterovirus D68 in two patients; all others were negative for potential etiologic agents.

All 23 patients with AFP also had anterior myelitis, inflammation of the grey matter of the spinal cord, which is characteristic of poliomyelitis. While the rate of AFP in California betweeen 1992-1998 was 1.4 cases per 100,000 children per year,  anterior myelitis was not described in any of 245 cases reviewed by CDC. However, poliovirus was ruled out as a cause in the 19 individuals who could be tested.

The cause of AFP is often difficult to determine because there infectious and non-infectious etiologies. Only 2 of the 19 clinical specimens met CDC guidelines for poliovirus detection (two stool specimens collected ≥24 hours apart and <14 days after symptom onset) and the others were likely taken too late to detect the presence of virus. The finding of enterovirus D68 in two of the samples is difficult to interpret, as the virus was detected in respiratory specimens and could have been a coincidental infection.

This investigation began with a request from a San Francisco area physician to the California State Department of Public Health to determine whether poliovirus was present in a 29 year old male with AFP and anterior myelitis. Subsequently this department posted alerts for AFP with anterior myelitis to  local health departments, and it is from the cases submitted that the 23 were drawn. Therefore the number of cases of AFP with anterior myelitis might be a consequence of this surveillance.

We are left with the unsatisfying conclusion that these 23 cases of AFP with anterior myelitis were either caused by an undetected infectious agent, or by something else.

*Defined by CDC as “at least one limb consistent with anterior myelitis, as indicated by neuroimaging of the spine or electrodiagnostic studies (e.g., nerve conduction studies and electromyography), and with no known alternative etiology”.

TWiV 305: Rhymes with shinola

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On episode #305 of the science show This Week in Virology, Vincent, Alan, and Kathy continue their coverage of the Ebola virus outbreak in West Africa, with a discussion of case fatality ratio, reproductive index, a conspiracy theory, and spread of the virus to the United States.

You can find TWiV #305 at www.microbe.tv/twiv.

An outbreak of enterovirus 68

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Enterovirus
EV-A71 by Jason Roberts

During the winter of 1962 in California, a new virus was isolated from the oropharynx of 4 children who had been hospitalized with respiratory disease that included pneumonia and bronchiolitis. On the basis of its physical, chemical, and biological properties, the virus was classified as an enterovirus in the picornavirus family. Subsequently named enterovirus D68, it has been rarely reported in the United States (there were 79 isolations from 2009-2013). Towards the end of August 2014, an outbreak of severe respiratory disease associated with EV-D68 emerged in Kansas and Illinois.

Hospitals in Kansas City, Missouri, and Chicago, Illinois reported to the CDC an increase in the number of patients hospitalized with severe respiratory illness. EV-D68 was subsequently identified by polymerase chain reaction and nucleotide sequencing in 19/22 and 11/14 nasopharyngeal specimens from Kansas City and Chicago, respectively. Median ages of the patients were 4 and 5 years in the two cities, and most were admitted to the pediatric intensive care units due to respiratory distress. Other states have reported increases in cases of severe respiratory illness, and these are being investigated at CDC to determine if they are also associated with EV-D68.

There is no vaccine to prevent EV-D68 infection, nor is antiviral therapy available to treat infected patients. Current treatment is supportive to assist breathing; in a healthy individual the infection will resolve within a week. In the current outbreak no fatalities have been reported.

EV-D68 has been previously associated with mild to severe respiratory illness and is known to cause clusters of infections. It is not clear why there has been a sudden increase in the number of cases in the US. According to Mark Pallansch, Director of the Division of Viral Diseases at CDC, “our ability to find and detect the virus has improved to the point where we may now be recognizing more frequently what has always occurred in the past. So a lot of these techniques are now being applied more routinely both at the CDC but also at state health departments.” (Source: NPR).

I am sure that the nucleotide sequence of the EV-D68 virus isolated from these patients will reveal differences with previous strains. However whether or not those changes have anything to do with the increased number of isolations in the US will be very difficult to determine, especially as there is no animal model for EV-D68 respiratory disease.

Although how EV-D68 is transmitted has not been well studied, the virus can be detected in respiratory secretions (saliva, nasal mucus, sputum) and is therefore likely to spread from person to person by coughing, sneezing, or touching contaminated surfaces. The virus has been isolated from some of the children in California with acute flaccid paralysis, and there is at least one report of its association with central nervous system disease. In this case viral nucleic acids were detected in the cerebrospinal fluid. EV-D68 probably does not replicate in the human intestinal tract because the virus is inactivated by low pH.

Readers might wonder why a virus that causes respiratory illness is called an enterovirus. This nomenclature is largely historical: poliovirus, which replicates in the enteric tract, was the prototype member of this genus. Other viruses, including Coxsackieviruses and echoviruses, were added to the genus based on their physical and chemical properties. However soon it became apparent that many of these viruses could also replicate in the respiratory tract. Years later the rhinoviruses, which do not replicate in the enteric tract, were added to the enterovirus genus based on nucleotide sequence comparisons. While it was decided to keep the name ‘enterovirus’ for this group of viruses, it is certainly confusing and I would argue that it should be replaced by a more descriptive name.