VP1 is the primary determinant of neuropathogenesis in a mouse model of enterovirus D68 acute flaccid myelitis.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.

Circulation of enterovirus A71 during 2019-2020, Marseille, France.

Enteroviruses A71 (EVs-A71) are known to cause serious neurological infections, especially in the pediatric population. We report here eight cases of EV-A71 infection diagnosed in Marseille over the past 2 years (seven cases in 2019 and one case in 2020). Only children under 5 years of age were affected, including one case of acute flaccid paralysis. Viral RNA was detected by RT-PCR in peripheral samples for all cases (feces and upper respiratory samples). Phylogenetic analyses based on VP1 and 2C3C coding regions revealed that all these cases of EV-A71 infection were caused by viruses belonging to the subgenogroup C1 that currently circulates in Europe and that these viruses are genetically closed to other EVs-A71 recently detected in European countries. These data therefore reinforce the usefulness of the enterovirus surveillance network and the need for systematic screening for EV-A71 in case of an enteroviral infection. This study therefore suggests that the systematic screening for EV-A71 in case of enteroviral infection could provide additional data for enterovirus surveillance networks.

IAPV-Induced Paralytic Symptoms Associated with Tachypnea via Impaired Tracheal System Function.

Although it had been reported that Israeli acute paralysis virus (IAPV) can cause systemic infection in honey bees, little is known about how it establishes this infection and results in the typical symptoms, paralysis and trembling. Here, we used our previously constructed IAPV infectious clone to investigate viral loads in different tissues of honey bees and further identify the relation between tissue tropism and paralytic symptoms. Our results showed that tracheae showed a greater concentration of viral abundance than other tissues. The abundance of viral protein in the tracheae was positively associated with viral titers, and was further confirmed by immunological and ultrastructural evidence. Furthermore, higher viral loads in tracheae induced remarkable down-regulation of succinate dehydrogenase and cytochrome c oxidase genes, and progressed to causing respiratory failure of honey bees, resulting in the appearance of typical symptoms, paralysis and body trembling. Our results showed that paralysis symptoms or trembling was actually to mitigate tachypnea induced by IAPV infection due to the impairment of honey bee tracheae, and revealed a direct causal link between paralysis symptoms and tissue tropism. These findings provide new insights into the understanding of the underlying mechanism of paralysis symptoms of honey bees after viral infection and have implications for viral disease prevention and specific therapeutics in practice.

An epidemiological analysis of Acute Flaccid Paralysis (AFP) surveillance in Kenya, 2016 to 2018.

BACKGROUND: The poliovirus has been targeted for eradication since 1988. Kenya reported its last case of indigenous Wild Poliovirus (WPV) in 1984 but suffered from an outbreak of circulating Vaccine-derived Poliovirus type 2 (cVDPV2) in 2018. We aimed to describe Kenya's polio surveillance performance 2016-2018 using WHO recommended polio surveillance standards. METHODS: Retrospective secondary data analysis was conducted using Kenyan AFP surveillance case-based database from 2016 to 2018. Analyses were carried out using Epi-Info statistical software (version 7) and mapping was done using Quantum Geographic Information System (GIS) (version 3.4.1). RESULTS: Kenya reported 1706 cases of AFP from 2016 to 2018. None of the cases were confirmed as poliomyelitis. However, 23 (1.35%) were classified as polio compatible. Children under 5 years accounted for 1085 (63.6%) cases, 937 (55.0%) cases were boys, and 1503 (88.1%) cases had received three or more doses of Oral Polio Vaccine (OPV). AFP detection rate substantially increased over the years; however, the prolonged health workers strike in 2017 negatively affected key surveillance activities. The mean Non-Polio (NP-AFP) rate during the study period was 2.87/ 100,000 children under 15 years, and two adequate specimens were collected for 1512 (88.6%) AFP cases. Cumulatively, 31 (66.0%) counties surpassed target for both WHO recommended AFP quality indicators. CONCLUSIONS: The performance of Kenya's AFP surveillance system surpassed the minimum WHO recommended targets for both non-polio AFP rate and stool adequacy during the period studied. In order to strengthen the country's polio free status, health worker's awareness on AFP surveillance and active case search should be strengthened in least performing counties to improve case detection. Similar analyses should be done at the sub-county level to uncover underperformance that might have been hidden by county level analysis.

A mouse model of paralytic myelitis caused by enterovirus D68.

In 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children coincident with a nationwide outbreak of enterovirus D68 (EV-D68) respiratory disease. Up to half of the 2014 AFM patients had EV-D68 RNA detected by RT-PCR in their respiratory secretions, although EV-D68 was only detected in cerebrospinal fluid (CSF) from one 2014 AFM patient. Given previously described molecular and epidemiologic associations between EV-D68 and AFM, we sought to develop an animal model by screening seven EV-D68 strains for the ability to induce neurological disease in neonatal mice. We found that four EV-D68 strains from the 2014 outbreak (out of five tested) produced a paralytic disease in mice resembling human AFM. The remaining 2014 strain, as well as 1962 prototype EV-D68 strains Fermon and Rhyne, did not produce, or rarely produced, paralysis in mice. In-depth examination of the paralysis caused by a representative 2014 strain, MO/14-18947, revealed infectious virus, virion particles, and viral genome in the spinal cords of paralyzed mice. Paralysis was elicited in mice following intramuscular, intracerebral, intraperitoneal, and intranasal infection, in descending frequency, and was associated with infection and loss of motor neurons in the anterior horns of spinal cord segments corresponding to paralyzed limbs. Virus isolated from spinal cords of infected mice transmitted disease when injected into naïve mice, fulfilling Koch's postulates in this model. Finally, we found that EV-D68 immune sera, but not normal mouse sera, protected mice from development of paralysis and death when administered prior to viral challenge. These studies establish an experimental model to study EV-D68-induced myelitis and to better understand disease pathogenesis and develop potential therapies.

Molecular characterization of enterovirus-A71 in children with acute flaccid paralysis in the Philippines.

BACKGROUND: Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study. METHODS: A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71-positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis. RESULTS: Nine EV-A71 isolates detected in 2000 (n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines. CONCLUSION: The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains.

Pathogenicity of egg-type duck-origin isolate of Tembusu virus in Pekin ducklings.

BACKGROUND: Tembusu virus (TMUV) usually affects adult ducks, causing a severe drop of egg production. It has also been shown to be pathogenic in commercial Pekin ducklings below 7 weeks of age. Here, we report a TMUV-caused neurological disease in young egg-type ducklings and the pathogenicity of the egg-type duck-origin TMUV isolates in meat-type Pekin ducklings. RESULTS: The disease occurred in 25 to 40-day-old Jinding ducklings in China, and was characterized by paralysis. Gross lesions were lacking and microscopic lesions appeared chiefly in brain and spleen. Inoculation in embryonated duck eggs resulted in isolation of TMUV Y and GL. The clinical signs and microscopic lesions observed in the spontaneously infected egg-type ducks were repeated in Pekin ducklings by experimental infection. Notably, both Y and GL strains caused 100% mortality in the case of 2-day-old inoculation by intracerebral route. High mortalities (80 and 70%) also occurred following infection of the Y virus at 2 days of age by intramuscular route and at 9 days of age by intracerebral route. CONCLUSIONS: These findings demonstrate that the egg-type duck-origin TMUVs exhibit high pathogenicity in Pekin ducklings, and that the severity of the disease in ducklings is dependent on the infection route and the age of birds at the time of infection. The availability of the highly pathogenic TMUV strains provides a useful material with which to begin investigations into the molecular basis of TMUV pathogenicity in ducks.

Hospital discharges-based search of acute flaccid paralysis cases 2007-2016 in Italy and comparison with the National Surveillance System for monitoring the risk of polio reintroduction.

BACKGROUND: Acute flaccid paralysis (AFP) surveillance has been adopted globally as a key strategy for monitoring the progress of the polio eradication initiative. Hereby, to evaluate the completeness of the ascertainment of AFP cases in Italy, a hospital-discharges based search was carried out. METHODS: AFP cases occurring between 2007 and 2016 among children under 15 years of age were searched in the Italian Hospital Discharge Records (HDR) database using specific ICD-9-CM diagnostic codes. AFP cases identified between 2015 and 2016 were then compared with those notified to the National Surveillance System (NSS). RESULTS: Over a 10-year period, 4163 hospital discharges with diagnosis of AFP were reported in Italy. Among these, 956 (23.0%) were acute infective polyneuritis, 1803 (43.3%) myopathy, and 1408 (33.8%) encephalitis, myelitis and encephalomyelitis. During the study period, a decreasing trend was observed for all diagnoses and overall the annual incidence rate (IR) declined from 5.5 to 4.5 per 100,000 children. Comparing NSS with HDR data in 2015-2016, we found a remarkable underreporting, being AFP cases from NSS only 14% of those recorded in HDR. In particular, the acute infective polyneuritis cases reported to NSS accounted for 42.6% of those detected in HDR, while only 0.9% of myopathy cases and 13.1% of encephalitis/myelitis/encephalomyelitis cases have been notified to NSS. The highest AFP IRs per 100,000 children calculated on HDR data were identified in Liguria (17.4), Sicily (5.7), and Veneto (5.1) Regions; regarding the AFP notified to the NSS, 11 out of 21 Regions failed to reach the number of expected cases (based on 1/100,000 rate), and the highest discrepancies were observed in the Northern Regions. Overall, the national AFP rate was equal to 0.6, therefore did not reach the target value. CONCLUSIONS: AFP surveillance data are the final measure of a country's progress towards polio eradication. The historical data obtained by the HDR have been useful to assess the completeness of the notification data and to identify the Regions with a low AFP ascertainment rate in order to improve the national surveillance system.

An increase in reports of acute flaccid paralysis (AFP) in the United Kingdom, 1 January 2018-21 January 2019: early findings.

During 2018, the United Kingdom experienced an increase in reports of cases of acute flaccid paralysis (AFP). As at 21 January 2019, 40 cases had been identified with a peak in October 2018. The increase was temporally associated with an upsurge in enterovirus (EV) D68 activity. Enterovirus was detected in 15 cases, mainly from respiratory tract samples; nine were typed as EV-D68. A national task force has been established and investigations are ongoing.

Clinical Features of Acute Flaccid Myelitis Temporally Associated With an Enterovirus D68 Outbreak: Results of a Nationwide Survey of Acute Flaccid Paralysis in Japan, August-December 2015.

Background: Acute flaccid myelitis (AFM) is an acute flaccid paralysis syndrome with spinal motor neuron involvement of unknown etiology. We investigated the characteristics and prognostic factors of AFM clusters coincident with an enterovirus D68 (EV-D68) outbreak in Japan during autumn 2015. Methods: An AFM case series study was conducted following a nationwide survey from August to December 2015. Radiographic and neurophysiologic data were subjected to centralized review, and virology studies were conducted for available specimens. Results: Fifty-nine AFM cases (58 definite, 1 probable) were identified, including 55 children and 4 adults (median age, 4.4 years). The AFM epidemic curve showed strong temporal correlation with EV-D68 detection from pathogen surveillance, but not with other pathogens. EV-D68 was detected in 9 patients: 5 in nasopharyngeal, 2 in stool, 1 in cerebrospinal fluid (adult case), and 1 in tracheal aspiration, nasopharyngeal, and serum samples (a pediatric case with preceding steroid usage). Cases exhibited heterogeneous paralysis patterns from 1- to 4-limb involvement, but all definite cases had longitudinal spinal gray matter lesions on magnetic resonance imaging (median, 20 spinal segments). Cerebrospinal fluid pleocytosis was observed in 50 of 59 cases (85%), and 8 of 29 (28%) were positive for antiganglioside antibodies, as frequently observed in Guillain-Barré syndrome. Fifty-two patients showed variable residual weakness at follow-up. Good prognostic factors included a pretreatment manual muscle strength test unit score >3, normal F-wave persistence, and EV-D68-negative status. Conclusions: EV-D68 may be one of the causative agents for AFM, while host susceptibility factors such as immune response could contribute to AFM development.

Outcomes in Patients With Severe West Nile Neuroinvasive Disease.

OBJECTIVE: To assess the long-term outcomes of patients hospitalized with severe West Nile neuroinvasive disease. DESIGN: Retrospective cohort. SETTING: Patients admitted to a referral center (Saint Mary's Hospital, Mayo Clinic). PARTICIPANTS: Twenty-six patients with West Nile neuroinvasive disease were identified by retrospective search of electronic database of Saint Mary's Hospital from January 1999 to November 2016. INTERVENTIONS: Retrospective electronic medical records review and prospective telephone follow-up. MEASUREMENTS AND MAIN RESULTS: Functional disability and cognitive outcomes were evaluated with the modified Rankin Scale and the Telephone Interview for Cognitive Status scores. Data on the time that the patient returned home after the hospitalization for West Nile neuroinvasive disease and the time of return to work were also collected. We identified 26 patients (81% males), 59 ± 17 years old. After a median hospital stay of 14.5 days (3-126), four patients died and 90% of survivors had a modified Rankin Scale of 3-5. Two additional patients died, and 80% of survivors had a modified Rankin Scale of 0-2 after a median follow-up of 73 months (1-144). Seven patients had cognitive impairment, which was severe in two of them. The combination of encephalitis and acute flaccid paralysis at presentation was associated with lower likelihood of returning home within 1 month after discharge (p < 0.01). Patients who required mechanical ventilation were more likely to have a modified Rankin Scale of 3-5 at last follow-up (p = 0.03), less likely to return home within 1 month of discharge (p < 0.01), less likely to return to their jobs (p < 0.01), and showed a trend toward having cognitive impairment (p = 0.05). CONCLUSIONS: Despite having poor outcomes at discharge, most West Nile neuroinvasive disease survivors with severe early disability can recover functional independence in the long term, justifying aggressive support during the acute phase and extensive rehabilitation efforts.

Vaccine-associated paralytic poliomyelitis in a patient with acute lymphocytic leukemia.

We report a case of vaccine-associated paralytic poliomyelitis (VAPP) in an immunocompromised patient with acute lymphocytic leukemia who was initially diagnosed with aseptic meningitis. Isolation of Sabin-like type 1 poliovirus from the patient's cerebrospinal fluid made this a case of vaccine-related poliovirus (VRPV) infection. The patient developed paralysis and respiratory distress and deceased a few months after onset of paralysis with respiratory failure. This tragic case report highlights the emergence of VAPP and indicates the importance of timely diagnosis of VRPV infections to improve clinical management of VRPV-infected patients and to prevent the devastating consequences of silent introduction of VRPVs in treatment wards and eventually in the society.

Population sensitivity of acute flaccid paralysis and environmental surveillance for serotype 1 poliovirus in Pakistan: an observational study.

BACKGROUND: To support poliomyelitis eradication in Pakistan, environmental surveillance (ES) of wastewater has been expanded alongside surveillance for acute flaccid paralysis (AFP). ES is a relatively new method of surveillance, and the population sensitivity of detecting poliovirus within endemic settings requires estimation. METHODS: Data for wild serotype 1 poliovirus from AFP and ES from January 2011 to September 2015 from 14 districts in Pakistan were analysed using a multi-state model framework. This framework was used to estimate the sensitivity of poliovirus detection from each surveillance source and parameters such as the duration of infection within a community. RESULTS: The location and timing of poliomyelitis cases showed spatial and temporal variability. The sensitivity of AFP surveillance to detect serotype 1 poliovirus infection in a district and its neighbours per month was on average 30.0% (95% CI 24.8-35.8) and increased with the incidence of poliomyelitis cases. The average population sensitivity of a single environmental sample was 59.4% (95% CI 55.4-63.0), with significant variation in site-specific estimates (median varied from 33.3-79.2%). The combined population sensitivity of environmental and AFP surveillance in a given month was on average 98.1% (95% CI 97.2-98.7), assuming four samples per month for each site. CONCLUSIONS: ES can be a highly sensitive supplement to AFP surveillance in areas with converging sewage systems. As ES for poliovirus is expanded, it will be important to identify factors associated with variation in site sensitivity, leading to improved site selection and surveillance system performance.

Characteristics of wild polio virus outbreak investigation and response in Ethiopia in 2013-2014: implications for prevention of outbreaks due to importations.

BACKGROUND: Ethiopia joined the Global Polio Eradication Initiative (GPEI) in 1996, and by the end of December 2001 circulation of indigenous Wild Polio Virus (WPV) had been interrupted. Nonetheless, the country experienced multiple importations during 2004-2008, and in 2013. We characterize the 2013 outbreak investigations and response activities, and document lessons learned. METHOD: The data were pulled from different field investigation reports and from the national surveillance database for Acute Flaccid Paralysis (AFP). RESULTS: In 2013, a WPV1 outbreak was confirmed following importation in Dollo zone of the Somali region, which affected three Woredas (Warder, Geladi and Bokh). Between July 10, 2013, and January 5, 2014, there were 10 children paralyzed due to WPV1 infection. The majorities (7 of 10) were male and below 5 years of age, and 7 of 10 cases was not vaccinated, and 72% (92/129) of < 5 years of old children living in close proximity with WPV cases had zero doses of oral polio vaccine (OPV). The travel history of the cases showed that seven of the 10 cases had contact with someone who had traveled or had a travel history prior to the onset of paralysis. Underserved and inaccessibility of routine immunization service, suboptimal surveillance sensitivity, poor quality and inadequate supplemental immunization were the most crucial gaps identified during the outbreak investigations. CONCLUSION: Prior to the 2013 outbreak, Ethiopia experienced multiple imported polio outbreaks following the interruption of indigenous WPV in December 2001. The 2013 outbreak erupted due to massive population movement and was fueled by low population immunity as a result of low routine immunization and supplemental Immunization coverage and quality. In order to avert future outbreaks, it is critical that surveillance sensitivity be improved by establishing community-based surveillance systems and by assigning surveillance focal points at all level particularly in border areas. In addition, it is vital to set up in hard to reach areas a functional immunization service delivery system using the "Reaching Every Child" approach, including periodic routine immunization intensification and supplemental immunization activities.

Paralytic rabies in a goat.

BACKGROUND: Paralytic form of rabies is frequent in cattle in Latin America, but it is uncommon in goats. There are few clinical reports on furious rabies affecting goats, and the sporadic cases of rabid goats from surveillance programs worldwide lack clinical data. Furthermore, few studies reported the cerebrospinal fluid findings in rabid livestock. CASE PRESENTATION: On a farm in Midwestern Brazil, six of 47 Saanen goats died within one week. No vaccination protocols were implemented on the farm and the owner stated bat bites history on the livestock. Although rabies is endemic in Brazil, livestock vaccination is not mandatory. One 1-year-old buck was evaluated and showed non-specific clinical signs evolving within 12-h to nervous signs. Cerebrospinal fluid analysis revealed mononuclear pleocytosis, hyperproteinemia and high glucose levels. At necropsy, no gross lesions were present. Microscopically, discrete to moderate perivascular lymphoplasmacytic cuffing in gray and white matter, neuronal necrosis, neuronophagia, and mononuclear ganglioneuritis was observed in the brainstem and cervical spinal cord. Immunohistochemistry revealed strong anti-rabies virus immunostaining. Fresh central nervous system samples were positive for rabies in direct fluorescent antibody test (dFAT) and mouse intracerebral inoculation test (MIT). Exposed livestock recommendations included immediate vaccination, a strict isolation period of 90 days, and booster vaccinations during the third and eighth weeks. CONCLUSION: IHC revealed the widespread distribution of rabies virus antigen in the goat's CNS, contrasting the discrete pathological changes. In this goat, definitive diagnosis of paralytic rabies was obtained through the association of epidemiological, clinical, laboratorial, pathological findings (histology and IHC) and gold standard confirmatory tests (dFAT and MIT).

Molecular diversity and biennial circulation of enterovirus D68: a systematic screening study in Lyon, France, 2010 to 2016.

BackgroundUnderstanding enterovirus D68 (EV-D68) circulation patterns as well as risk factors for severe respiratory and neurological illness is important for developing preventive strategies. Methods: Between 2010 and 2016, 11,132 respiratory specimens from hospitalised patients in Lyon, France, were screened for EV-D68 by PCR. Phylogenetic relationships of the viral-protein-1 sequences were reconstructed using maximum-likelihood and Bayesian-Markov-Chain-Monte-Carlo approaches. Results: Overall, 171 infections with a biennial pattern were detected, including seven, one, 55, none, 42, one and 65 cases annually during 2010-16. Children (< 16 years-old; n = 150) were mostly affected and 71% (n = 121) of the total patients were under 5 years-old. In 146 patients with medical reviews, 73% (n = 107) presented with acute respiratory distress. Among paediatric patients with medical reviews (n = 133), 55% (n=73) had an asthma/wheezing history, while among adults (n = 13), 11 had underlying diseases. In total, 45 patients had severe infections and 28 patients needed intensive care unit stays. No acute flaccid myelitis (AFM) was detected. We found genotypes A, B1, B2 B3 and D circulating, and no associations between these and clinical presentations. During the study, new genotypes continuously emerged, being replaced over time. We estimated that ancestors of currently circulating genotypes emerged in the late-1990s to 2010. Rises of the EV-D68 effective population size in Lyon coincided with infection upsurges. Phylogenetic analyses showed ongoing diversification of EV-D68 worldwide, coinciding with more infections in recent years and increases of reported AFM paediatric cases. Conclusions: Reinforcement of diagnostic capacities and clinical-based surveillance of EV-D68 infections is needed in Europe to assess the EV-D68 burden.

Using Acute Flaccid Paralysis Surveillance as a Platform for Vaccine-Preventable Disease Surveillance.

Surveillance for acute flaccid paralysis (AFP) is a fundamental cornerstone of the global polio eradication initiative (GPEI). Active surveillance (with visits to health facilities) is a critical strategy of AFP surveillance systems for highly sensitive and timely detection of cases. Because of the extensive resources devoted to AFP surveillance, multiple opportunities exist for additional diseases to be added using GPEI assets, particularly because there is generally 1 district officer responsible for all disease surveillance. For this reason, integrated surveillance has become a standard practice in many countries, ranging from adding surveillance for measles and rubella to integrated disease surveillance for outbreak-prone diseases (integrated disease surveillance and response). This report outlines the current level of disease surveillance integration in 3 countries (Nepal, India, and Nigeria) and proposes that resources continue for long-term maintenance in resource-poor countries of AFP surveillance as a platform for surveillance of vaccine-preventable diseases and other outbreak-prone diseases.

Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis.

Background: Enterovirus D68 (EV-D68)-associated acute flaccid myelitis (AFM) is a devastating neurological disease for which there are no treatments of proven efficacy. The unpredictable temporal and geographic distribution of cases and the rarity of the disease make it unlikely that data from randomized controlled trials will be available to guide therapeutic decisions. We evaluated the following 3 widely used empirical therapies for the 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. Methods: Neonatal mice were injected intramuscularly with a human 2014 EV-D68 isolate that reliably induces paralysis in mice due to infection and loss of spinal cord motor neurons. Mice receiving treatments were evaluated for motor impairment, mortality, and spinal cord viral load. Results: hIVIG, which contained neutralizing antibodies to 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. Conclusion: Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans and establish this model as a useful system for evaluating other potential therapies.

Protective Effects of Glutamine Antagonist 6-Diazo-5-Oxo-l-Norleucine in Mice with Alphavirus Encephalomyelitis.

UNLABELLED: Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease. IMPORTANCE: Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease.