Susceptibility and cytokine responses of human neuronal cells to multiple circulating EV-A71 genotypes in India.

Enterovirus-A71 (EV-A71) associated Hand, foot and mouth disease (HFMD) is a highly contagious viral infection affecting children in Asia-Pacific region and has become a major threat to public health. Although several EV-A71 genotypes (C, D, and G) were isolated in India in recent years, no recognizable outbreak of EV-A71 caused HFMD, Acute Flaccid paralysis (AFP) or encephalitis have been reported so far. It is essential to study the pathogenicity or cell tropism of these Indian isolates in order to understand their tendency to cause disease. We investigated the susceptibility and cytokine responses of indigenous EV-A71 genotypes (D and G) isolated from cases of AFP and genotype C viruses isolated from cases of HFMD and encephalitis, in human cells in-vitro. Although all three EV-A71 genotypes could infect and replicate in human muscle and neuronal cells, the genotype D virus showed a delayed response in human neuronal cells. Quantification of cytokine secretion in response to these isolates followed by confirmation with gene expression assays in human neuronal cells revealed significantly higher secretion of pro-inflammatory cytokines TNF-α IL-8, IL-6, IP-10 (p < 0.001) in G genotype infected cells as compared to pathogenic C genotypes whereas the genotype D virus could not induce any of the inflammatory cytokines. These findings will help to better understand the host response to indigenous EV-A71 genotypes for management of future EV-A71 outbreaks in India, if any.

Acute transverse myelitis after SARS-CoV-2 infection: a rare complicated case of rapid onset paraplegia.

A 63-year-old Caucasian male, known case of controlled type 2 diabetes, chronic renal failure, and ischemic heart disease, was presented with weakness and loss of movement in lower limbs, an absent sensation from the chest below, constipation, and urinary retention. About 4 days before these symptoms, he experienced a flu-like syndrome. Suspicious for COVID-19, his nasopharyngeal specimen's reverse transcription-polymerase chain reaction (RT-PCR) resulted positive. Chest X-ray and HRCT demonstrated severe pulmonary involvement. Immediately, he was admitted to the emergency ward, and the treatment was started according to the national COVID-19 treatment protocol. Subsequently, diagnostic measures were taken to investigate the patient's non-heterogeneous peripheral (spinal) neuromuscular manifestations. Brain CT scan and MRI were normal, but spinal MRI with gadolinium contrast showed extensive increased T2 signal involving central gray matter and dorsal columns, extended from C7 to T12 with linear enhancement in the sagittal plane, posteriorly within the mid and lower thoracic cord. The CSF specimen demonstrated pleocytosis, positive RT-PCR for SARS-CoV-2, and elevated IgG index. Clinical presentation, MRI, CSF, and laboratory findings prioritized the acute transverse myelitis (ATM) as a probable complication of COVID-19 infection over other differential diagnoses. Intravenous methylprednisolone and, subsequently, IV human immunoglobulin were added to the treatment regimen. In the end, the complete resolution of dysesthesia, urinary retention, and constipation were achieved. After continuous and extended respiratory and motor rehabilitation programs, he was discharged asymptomatic.

Australian National Enterovirus Reference Laboratory annual report, 2015.

Australia conducts surveillance for cases of acute flaccid paralysis (AFP) in children less than 15 years as recommended by the World Health Organization (WHO) as the main method to monitor its polio-free status. Cases of AFP in children are notified to the Australian Paediatric Surveillance Unit or the Paediatric Active Enhanced Disease Surveillance System and faecal specimens are referred for virological investigation to the National Enterovirus Reference Laboratory. In 2015, no cases of poliomyelitis were reported from clinical surveillance and Australia reported 1.2 non-polio AFP cases per 100,000 children, meeting the WHO performance criterion for a sensitive surveillance system. Two non-polio enteroviruses, enterovirus A71 and coxsackievirus B3, were identified from clinical specimens collected from AFP cases. Australia complements the clinical surveillance program with enterovirus and environmental surveillance for poliovirus. Two Sabin-like polioviruses were isolated from sewage collected in Melbourne in 2015, which would have been imported from a country that uses the oral polio vaccine. The global eradication of wild poliovirus type 2 was certified in 2015 and Sabin poliovirus type 2 will be withdrawn from oral polio vaccine in April 2016. Laboratory containment of all remaining wild and vaccine strains of poliovirus type 2 will occur in 2016 and the National Enterovirus Reference Laboratory was designated as a polio essential facility. Globally, in 2015, 74 cases of polio were reported, only in the two remaining countries endemic for wild poliovirus: Afghanistan and Pakistan. This is the lowest number reported since the global polio eradication program was initiated.

Australian National Enterovirus Reference Laboratory annual report, 2016.

Australia monitors its polio-free status by conducting surveillance for cases of acute flaccid paralysis (AFP) in children less than 15 years of age, as recommended by the World Health Organization (WHO). Cases of AFP in children are notified to the Australian Paediatric Surveillance Unit or the Paediatric Active Enhanced Disease Surveillance System and faecal specimens are referred for virological investigation to the National Enterovirus Reference Laboratory. In 2016, no cases of poliomyelitis were reported from clinical surveillance and Australia reported 1.38 non-polio AFP cases per 100,000 children, meeting the WHO performance criterion for a sensitive surveillance system. Several non-polio enteroviruses, coxsackievirus A6, enterovirus A71, enterovirus A74 and enterovirus D68, were identified from clinical specimens collected from AFP cases. The global withdrawal of Sabin poliovirus type 2 from oral polio vaccine occurred in April 2016. This event represents the start of the polio endgame with an increased focus on the laboratory containment of all remaining wild and vaccine strains of poliovirus type 2. The National Enterovirus Reference Laboratory was designated as a polio essential facility as part of this process. In 2016, 37 cases of wild polio were reported with three countries remaining endemic: Afghanistan, Nigeria and Pakistan. Nigeria was declared polio-free in 2015, after 12 months without detection of wild poliovirus, but was reinstated as an endemic country after the reporting of four cases in August 2016. This is a salient reminder of the need to maintain sensitive surveillance for poliovirus until global eradication is certified.

Australian National Enterovirus Reference Laboratory annual report, 2017.

Australia monitors its polio-free status by conducting surveillance for cases of acute flaccid paralysis (AFP) in children less than 15 years of age, as recommended by the World Health Organization (WHO). Cases of AFP in children are notified to the Australian Paediatric Surveillance Unit or the Paediatric Active Enhanced Disease Surveillance System and faecal specimens are referred for virological investigation to the National Enterovirus Reference Laboratory. In 2017, no cases of poliomyelitis were reported from clinical surveillance and Australia reported 1.33 non-polio AFP cases per 100,000 children, meeting the WHO performance criterion for a sensitive surveillance system. Three non-polio enteroviruses, coxsackievirus B1, echovirus 11 and enterovirus A71, were identified from clinical specimens collected from AFP cases. Australia established enterovirus and environmental surveillance systems to complement the clinical system focussed on children and an ambiguous vaccine-derived poliovirus type 2 was isolated from sewage in Melbourne. In 2017, 22 cases of wild polio were reported with three countries remaining endemic: Afghanistan, Nigeria and Pakistan.

Australian National Enterovirus Reference Laboratory annual report, 2018.

Australia monitors its polio-free status by conducting surveillance for cases of AFP in children less than 15 years of age, as recommended by the WHO. Cases of AFP in children are notified to the Australian Paediatric Surveillance Unit or the Paediatric Active Enhanced Disease Surveillance System and faecal specimens are referred for virological investigation to the National Enterovirus Reference Laboratory. In 2018, no cases of poliomyelitis were reported from clinical surveillance and Australia reported 1.24 non-polio AFP cases per 100,000 children, meeting the WHO performance criterion for a sensitive surveillance system. Several non-polio enteroviruses, coxsackievirus A4, coxsackievirus B1, echovirus 9, echovirus 30, enterovirus D68 and enterovirus A71, were identified from clinical specimens collected from AFP cases. Australia also performs enterovirus and environmental surveillance to complement the clinical system focussed on children. In 2018, 33 cases of wild polio were reported with three countries remaining endemic: Afghanistan, Nigeria and Pakistan.

Multiple divergent Human mastadenovirus C co-circulating in mainland of China.

The human mastadenovirus C (HAdV-C) cause respiratory infections in children. Homologous recombination was clearly involved in the molecular evolution of HAdV-A, B, and D, but little is known about the molecular evolution of HAdV-C. From 2000 to 2016, 201 HAdV-C strains were collected from nine provinces covering six administrative regions of mainland of China via 3 existing surveillance programs, namely the febrile respiratory syndrome surveillance, the acute flaccid paralysis surveillance, and the hand, foot, and mouth disease surveillance system. The genes coding for the capsid protein (penton base, hexon, and fiber) of 201 HAdV-C strains were sequenced and compared with representative sequences publicly available. In addition, the whole genome sequence of 24 representative strains of HAdV-C was generated for further recombination analysis. Phylogenetic analysis of the penton base sequences of HAdV-C revealed six genetic groups (labelled as Px1-6), which showed that the penton base had more variation than previously thought. Based on the penton base, hexon, and fiber gene sequences, 16 new genetic patterns of HAdV-C circulating in mainland of China were identified in this study. Whole genome sequence analysis revealed frequent recombination events among HAdV-C genomes. This study is highly beneficial for case classification, tracking the transmission chain, and further epidemiological exploration of HAdV-C-related severe clinical diseases in the near future. Our data demonstrated that multiple newly divergent HAdV-C co-circulated across mainland China during the research period.

Metagenomic analysis identifies human adenovirus 31 in children with acute flaccid paralysis in Tunisia.

A variety of viruses can cause acute flaccid paralysis (AFP). However, the causative agent, sometimes, remains undetermined. Metagenomics helps in identifying viruses not diagnosed by conventional methods. Stool samples from AFP (n = 104) and non-AFP (n = 114) cases that tested enterovirus-negative by WHO standard methods were investigated. A metagenomics approach, first used on five pools of four samples each, revealed the presence of adenovirus sequences. Amplification in A549 cells and full-genome sequencing were used for complete virus identification and for designing a PCR assay to screen individual related samples. Metagenomic analysis showed that adenovirus sequences that were closely to the A31 and A61 genotypes were the most abundant. Two out of the corresponding 20 individual samples were found positive by PCR, and isolates were obtained in cell culture. Phylogenetic analysis based on complete genome sequences showed that the viruses belong to HAdV-A31 genotype (98-100% nucleotide sequence identity). PCR analysis of stool samples from all AFP and non-AFP cases revealed that a larger proportion of the positive samples were from AFP cases (17.3%) than from non-AFP cases (2.4%). These results open the way to studies aiming to test a possible role of HAdV-A31 in the pathogenesis of AFP.

Micro-planning for immunization in Kaduna State, Nigeria: Lessons learnt, 2017.

BACKGROUND: The OPV 3 coverage for Kaduna State, 12-23 months old children was 34.4%. The low OPV 3 coverage, due mainly to weak demand for routine antigens and the need to rapidly boost population immunity against the disabling Wild Polio Virus (WPV), led the Global Polio Eradication Initiatives (GPEI) to increase supplemental OPV campaigns in Kaduna State, despite the huge cost and great burden on personnel. The OPV campaigns, especially in high risk (low vaccine uptake, <80% OPV 3 coverage and high vaccines refusal rate) states of northern Nigeria with poliovirus transmission has resulted in overestimated denominators or target population, as the highest ever vaccinated is used to set OPV campaign targets. METHODS: We utilized a cross-sectional study that assessed the impacts and possible solutions to the challenges of overestimated denominators in immunization services planning, delivery and performance evaluation in Kaduna State, Nigeria. We used both descriptive and quantitative approaches. We enumerated households and obtained the target populations for routine immunization (<1 year), polio campaign (<5 years) and acute flaccid paralysis surveillance (<15 years). RESULTS: We found a significant difference in mean scores between the micro-planning and supplemental vaccination data on a number of <5 years (M = 102967, SD = 62405, micro-planning compared to M = 157716, SD = 72212, supplemental vaccination, p < 0.05). We also found a significant difference in mean scores between the micro-planning and projected census data on a number of <1 year (M = 26128, SD = 16828, micro-planning compared to M = 14154, SD = 4894, census, p < 0.05). CONCLUSION: Periodic household-based micro-planning, aided with the use of technology for validation remains a useful tool in addressing gaps in immunization planning, delivery and performance evaluation in developing countries, such as Nigeria with overestimated denominators.

Assessment of enteroviruses from sewage water and clinical samples during eradication phase of polio in North India.

BACKGROUND: The Enterovirus (EV) surveillance system is inadequate in densely populated cities in India. EV can be shed in feces for several weeks; these viruses are not easily inactivated and may persist in sewage for long periods. Surveillance and epidemiological study of EV-related disease is necessary. METHODS: In this study, we compare the EV found in sewage with clinically isolated samples. Tissue culture was used for isolation of the virus and serotype confirmed by enterovirus neutralization tests. RESULTS: We found positive cases for enterovirus from clinical and sewage samples and identified additional isolates as echovirus 9, 11, 25 & 30 by sequencing. CONCLUSION: There is a close relation among the serotypes of enterovirus shed in stools and isolated from the environment but few serotypes which were detected in sewage samples were not found clinically and the few which were detected clinically not found in sewage because some viruses are difficult to detect by the cell culture method.This study will be helpful for the researchers who are working on polio and nonpolio enterovirus especially in the countries which are struggling for polio eradication.

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.

Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria is geographically defined.

PURPOSE: We recently showed that enteroviruses (EVs) andenterovirus species C (EV-C) in particular were abundant in faecal samples from children who had been diagnosed with acute flaccid paralysis (AFP) in Nigeria but declared to be EV-free by the RD-L20B cell culture-based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples varies by geographical region. METHODOLOGY: One hundred and eight samples (i.e. 54 paired stool suspensions from 54 AFP cases) that had previously been confirmed to be negative for EVs by the WHO-recommended RD-L20B cell culture-based algorithm were analysed. The 108 samples were made into 54 pools (27 each from North-West and South-South Nigeria). All were subjected to RNA extraction, cDNA synthesis and the WHO-recommended semi-nested PCR assay and its modifications. All of the amplicons were sequenced, and the enteroviruses identified, using the enterovirus genotyping tool and phylogenetic analysis. RESULTS: EVs were detected in 16 (29.63 %) of the 54 samples that were screened and successfully identified in 14 (25.93 %). Of these, 10 were from North-West and 4 were from South-South Nigeria. One (7.14 %), 2 (14.29 %) and 11 (78.57 %) of the strains detected were EV-A, EV-B and EV-C, respectively. The 10 strains from North-West Nigeria included 7 EV types, namely CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from South-South Nigeria were E31, CV-A1, EV-C99 and EV-C116. CONCLUSION: The results of this study showed that the presence of EVs and consequently EV-Cs in AFP samples declared to be EV-free by the RD-L20B cell culture-based algorithm varies by geographical region in Nigeria.

Strengthening Acute Flaccid Paralysis Surveillance Through the Village Polio Volunteers Program in Somalia.

Background: Surveillance for cases of acute flaccid paralysis (AFP) is a key strategy adopted for the eradication of polio. Detection of poliovirus circulation is often predicated on the ability to identify AFP cases and test their stool specimens for poliovirus infection in a timely manner. The Village Polio Volunteers (VPV) program was established in 2013 in a bid to strengthen polio eradication activities in Somalia, including AFP surveillance, given the country's vulnerability to polio outbreaks. Methods: To assess the impact of the VPV program on AFP surveillance, we determined case counts, case-reporting sources, and nonpolio AFP rates in the years before and after program introduction (ie, 2011-2016). We also compared the stool specimen adequacy rates and timeliness of cases reported by VPVs to those reported by other sources. Results: In the years after program introduction, VPVs accounted for a high proportion of AFP cases reported in Somalia. AFP case counts rose from 148 cases in 2012, the year before program introduction, to 279 cases in 2015, when VPVs accounted for 40% of reported cases. Further, from 2012 to 2015, the nonpolio AFP rate improved from 2.8 to 4.8 cases per 100000 persons aged <15 years. Stool specimen adequacy rates have been consistently high, and AFP cases have been detected in a timelier manner since the program was introduced. Conclusions: Given the impact of the VPV program on improving AFP surveillance indicators in Somalia, similar community-based programs could play a crucial role in enhancing surveillance activities in countries with limited healthcare infrastructure.

Updates on the molecular epidemiology of Enterovirus D68 after installation of screening test among acute flaccid paralysis patients in Taiwan.

In respond to acute flaccid paralysis (AFP) in association with Enterovirus D68 (EV-D68) infection, Taiwan Centers for Disease Control began to screen EV-D68 infection among each AFP patient since July 2015 and detected the first case in August 2016. This article updated the molecular epidemiology trends of EV-D68 from the national surveillance data.

Genetic characterization of enterovirus A71 isolates from severe neurological cases in Poland.

The aim of this study was to report a minor outbreak of enterovirus A71 (EV-A71) infection in Poland and characterize isolates from cases of severe neurological infection detected in 2013 and 2016. Phylogenetic analysis revealed that Polish strains belonged to the C genogroup: C1, C2, and C4. Severe neurological manifestations as encephalitis or acute flaccid paralysis (AFP), were associated with all detected subgenogroups. The C2 subgenogroup was associated with the outbreak in Gdansk, with serious cases of AFP, myelitis, cerebellitis, encephalitis, but also with mild, sporadic cases of aseptic meningitis, in other Polish cities. Data from the study established relationships of EV-A71 from Poland with previously characterized strains and confirmed the importance of high quality enterovirus surveillance with international reach.

Non-polio Enterovirus detection with acute flaccid paralysis: A systematic review.

Acute flaccid paralysis (AFP), as defined by the World Health Organization (WHO), is characterized by an acute onset of limb weakness. In the post-polio era, other enterovirus (EV) serotypes associated with AFP may become more prominent. This study aims to collate the data on the non-polio enteroviruses (NPEV) associated with AFP. A systematic review of published case reports, case series, and surveillance studies of AFP from 1960 through 2017 was undertaken. Data were collected including the country of the study, number of specimens positive for NPEV and available clinical data. The majority of studies originated from Asia. In surveillance studies, EV 71 (a serotype of Enterovirus A) was the most commonly detected serotype with AFP, followed by Enterovirus B serotype echovirus 11 and then Enterovirus B serotype echovirus 11. In case studies and case reports, EV 71 and EV 68 (a serotype of Enterovirus D), were the most commonly detected NPEV. As poliovirus eradication continues, there is a need to ensure that AFP surveillance will also detect other potentially vaccine preventable viruses.

Haemorrhagic conversion of infectious myelitis in an immunocompromised patient.

A 28-year-old man recently diagnosed with HIV (CD4 19 cells/mm3, viral load 3.6 million copies/mL, not on highly active antiretroviral therapy on initial diagnosis at outside hospital), disseminated histoplasmosis, shingles and syphilis presented with paraplegia developing over 3 days. Spine MRI demonstrated a longitudinally extensive cord lesion extending from C3 to the tip of the conus. Brain MRI was consistent with meningoencephalitis. Cerebrospinal fluid findings were notable for positive varicella zoster virus (VZV) and cytomegalovirus (CMV) PCRs as well as a Venereal Disease Research Laboratory titre of 1:2. Patient was started on treatment for VZV and CMV meningoencephalitis, neurosyphilis and high-dose steroids for infectious myelitis. Repeat spine MRI demonstrated subacute intramedullary haemorrhage of the cervical cord. He was ultimately discharged to a skilled nursing facility for long-term intravenous antiviral therapy and rehabilitation. After 59 days in the hospital, his neurological exam remained grossly unchanged, with flaccid paraplegia and lack of sensation to fine touch in his lower extremities.

Outbreaks of Neuroinvasive Astrovirus Associated with Encephalomyelitis, Weakness, and Paralysis among Weaned Pigs, Hungary.

A large, highly prolific swine farm in Hungary had a 2-year history of neurologic disease among newly weaned (25- to 35-day-old) pigs, with clinical signs of posterior paraplegia and a high mortality rate. Affected pigs that were necropsied had encephalomyelitis and neural necrosis. Porcine astrovirus type 3 was identified by reverse transcription PCR and in situ hybridization in brain and spinal cord samples in 6 animals from this farm. Among tissues tested by quantitative RT-PCR, the highest viral loads were detected in brain stem and spinal cord. Similar porcine astrovirus type 3 was also detected in archived brain and spinal cord samples from another 2 geographically distant farms. Viral RNA was predominantly restricted to neurons, particularly in the brain stem, cerebellum (Purkinje cells), and cervical spinal cord. Astrovirus was generally undetectable in feces but present in respiratory samples, indicating a possible respiratory infection. Astrovirus could cause common, neuroinvasive epidemic disease.

Australian National Enterovirus Reference Laboratory annual report, 2014.

Following the World Health Organization (WHO) recommendation, Australia conducts surveillance for cases of acute flaccid paralysis (AFP) in children less than 15 years of age as the main method to monitor its polio-free status. Cases of AFP in children are notified to the Australian Paediatric Surveillance Unit or the Paediatric Active Enhanced Disease Surveillance System and faecal specimens are referred for virological investigation to the National Enterovirus Reference Laboratory. In 2014, no cases of poliomyelitis were reported from clinical surveillance and Australia reported 1.4 non-polio AFP cases per 100,000 children, meeting the WHO performance criterion for a sensitive surveillance system. Non-polio enteroviruses can also be associated with AFP and enterovirus A71 and echovirus types 6 and 7 were identified from clinical specimens from cases of AFP. Globally, 359 cases of polio were reported in 2014, with the 3 endemic countries, Afghanistan, Nigeria and Pakistan, accounting for 95% of the cases. In May 2014, the WHO declared the international spread of wild poliovirus to be a public health emergency of international concern and has since maintained recommendations for polio vaccination of travellers from countries reporting cases of wild polio.