Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

First description and phylogenetic analysis of coxsackie virus A non-polio enteroviruses and parechoviruses A in South Sudanese children.

Enteroviruses (EV) and parechoviruses A (PeV-A) are commonly circulating viruses able to cause severe disease. Surveillance studies from sub-Saharan Africa are limited and show high but variable infection rates and a high variation in genotypes. This is the first study to describe EV and PeV-A circulation in children in South Sudan. Of the fecal samples collected, 35% and 10% were positive for EV and PeV-A, respectively. A wide range of genotypes were found, including several rarely described EV and PeV-A types. Coxsackie virus A (CVA) EV-C types, particularly CVA13, were the most dominant EV types. The CVA13 types had a high diversity with the majority belonging to four different previously described clusters. PeV-A1 and -A14 were the most common PeV-A genotypes. A lack of representative data from our and other studies from sub-Saharan Africa demonstrates the need for more systematic surveillance of non-polio EV and PeV-A types in this region.

Parasitic, bacterial, viral, immune-mediated, metabolic and nutritional factors associated with nodding syndrome.

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus. Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect.

Focal lesions following intracerebral gene therapy for mucopolysaccharidosis IIIA.

OBJECTIVE: Mucopolysaccharidosis type IIIA (MPSIIIA) caused by recessive SGSH variants results in sulfamidase deficiency, leading to neurocognitive decline and death. No disease-modifying therapy is available. The AAVance gene therapy trial investigates AAVrh.10 overexpressing human sulfamidase (LYS-SAF302) delivered by intracerebral injection in children with MPSIIIA. Post-treatment MRI monitoring revealed lesions around injection sites. Investigations were initiated in one patient to determine the cause. METHODS: Clinical and MRI details were reviewed. Stereotactic needle biopsies of a lesion were performed; blood and CSF were sampled. All samples were used for viral studies. Immunohistochemistry, electron microscopy, and transcriptome analysis were performed on brain tissue of the patient and various controls. RESULTS: MRI revealed focal lesions around injection sites with onset from 3 months after therapy, progression until 7 months post therapy with subsequent stabilization and some regression. The patient had transient slight neurological signs and is following near-normal development. No evidence of viral or immunological/inflammatory cause was found. Immunohistochemistry showed immature oligodendrocytes and astrocytes, oligodendrocyte apoptosis, strong intracellular and extracellular sulfamidase expression and hardly detectable intracellular or extracellular heparan sulfate. No activation of the unfolded protein response was found. INTERPRETATION: Results suggest that intracerebral gene therapy with local sulfamidase overexpression leads to dysfunction of transduced cells close to injection sites, with extracellular spilling of lysosomal enzymes. This alters extracellular matrix composition, depletes heparan sulfate, impairs astrocyte and oligodendrocyte function, and causes cystic white matter degeneration at the site of highest gene expression. The AAVance trial results will reveal the potential benefit-risk ratio of this therapy.

Nodding Syndrome: Clinical Characteristics, Risks Factors, Access to Treatment, and Perceptions in the Greater Mundri Area, South Sudan.

We conducted a house-to-house survey in the Mundri, Western Equatoria state of South Sudan to investigate the clinical characteristics, risk factors, access to treatment and perceptions about nodding syndrome (NS). In total, 224 NS cases with median age of seizure onset of 10 years were identified. Head nodding only was reported in 50 (22.3%) cases, and head nodding plus other types of seizures in 174 (77.7%) cases. Wasting, stunted growth, delayed sexual development and speech and behavioral abnormalities were observed in 17 (23.6%), 16 (22.2%), 9 (17.3%), 14 (19.4%) and 4 (5.6%) cases, respectively. The consumption of rat meat, but not other bushmeat was associated with an increased risk of NS (OR 9.31, 95% CI 1.27-406.51). Children with NS were more likely to have taken ivermectin in the last 5 years (OR 2.40, 95% CI 1.33-4.43). NS cases were less likely to share a bedroom with other children (OR 0.06, 95% CI 0.02-0.16) or adults (OR 0.27, 95% CI 0.13-0.56). In conclusion, rat meat consumption is an unlikely risk factor for NS, and ivermectin intake was more common among NS cases than controls. Importantly, we documented that children with NS are stigmatized because of the misconception that NS is transmitted through direct contact.

Bacterial ribosomal RNA detection in cerebrospinal fluid using a viromics approach.

BACKGROUND: In patients with central nervous system (CNS) infections identification of the causative pathogen is important for treatment. Metagenomic next-generation sequencing techniques are increasingly being applied to identify causes of CNS infections, as they can detect any pathogen nucleic acid sequences present. Viromic techniques that enrich samples for virus particles prior to sequencing may simultaneously enrich ribosomes from bacterial pathogens, which are similar in size to small viruses. METHODS: We studied the performance of a viromic library preparation technique (VIDISCA) combined with low-depth IonTorrent sequencing (median ~ 25,000 reads per sample) for detection of ribosomal RNA from common pathogens, analyzing 89 cerebrospinal fluid samples from patients with culture proven bacterial meningitis. RESULTS: Sensitivity and specificity to Streptococcus pneumoniae (n = 24) before and after optimizing threshold parameters were 79% and 52%, then 88% and 90%. Corresponding values for Neisseria meningitidis (n = 22) were 73% and 93%, then 67% and 100%, Listeria monocytogenes (n = 24) 21% and 100%, then 27% and 100%, and Haemophilus influenzae (n = 18) 56% and 100%, then 71% and 100%. A higher total sequencing depth, no antibiotic treatment prior to lumbar puncture, increased disease severity, and higher c-reactive protein levels were associated with pathogen detection. CONCLUSION: We provide proof of principle that a viromic approach can be used to correctly identify bacterial ribosomal RNA in patients with bacterial meningitis. Further work should focus on increasing assay sensitivity, especially for problematic species (e.g. L. monocytogenes), as well as profiling additional pathogens. The technique is most suited to research settings and examination of idiopathic cases, rather than an acute clinical setting.

Recent infection with HCoV-OC43 may be associated with protection against SARS-CoV-2 infection.

Antibodies against seasonal human coronaviruses (HCoVs) are known to cross-react with SARS-CoV-2, but data on cross-protective effects of prior HCoV infections are conflicting. In a prospective cohort of healthcare workers (HCWs), we studied the association between seasonal HCoV (OC43, HKU1, 229E and NL63) nucleocapsid protein IgG and SARS-CoV-2 infection during the first pandemic wave in the Netherlands (March 2020 - June 2020), by 4-weekly serum sampling. HCW with HCoV-OC43 antibody levels in the highest quartile, were less likely to become SARS-CoV-2 seropositive when compared with those with lower levels (6/32, 18.8%, versus 42/97, 43.3%, respectively: p = 0.019; HR 0.37, 95% CI 0.16-0.88). We found no significant association with HCoV-OC43 spike protein IgG, or with antibodies against other HCoVs. Our results indicate that the high levels of HCoV-OC43-nucleocapsid antibodies, as an indicator of a recent infection, are associated with protection against SARS-CoV-2 infection; this supports and informs efforts to develop pancoronavirus vaccines.

Epidemiology of nodding syndrome in the Greater Mundri area, South Sudan: Prevalence, spatial pattern and environmental risk factors.

BACKGROUND: Nodding syndrome (NS) is a progressive neurological disease that has been described in several sub-Saharan African counties, but South Sudan is considered the most affected. However, knowledge about the exact burden and the epidemiological risk factors of NS in South Sudan is lacking. OBJECTIVE: To determine the prevalence, distribution and epidemiological risk factors of NS in the Greater Mundri area, the epicenter of NS in South Sudan. METHODS: A NS prevalence house-to-house survey was conducted in multiple villages between February 2018 and November 2019. Geographical distribution and clustering of NS cases was identified using spatial and binomial regression analysis. Epidemiological risk factors of NS were identified using univariate and multivariate models. RESULTS: Of the 22,411 persons surveyed in 92 villages, 607 (2.7%) persons with NS were identified, of which 114 (19%) were new-onset cases. The highest prevalence was found in Diko village with a prevalence of 13.7%. NS showed a significant spatial pattern with clustering of cases between adjacent households and along rivers. Risks factors for NS include all behaviors around rivers (drinking, cooking, handwashing and bathing) and exposure to poultry. On the other hand, ownership of mobile phone decreased the risk of NS. Many other factors, including prior ivermectin treatment and internal displacement were not associated with NS. CONCLUSION: Our study demonstrates a very high burden of the NS disease in the Greater Mundri area, strengthens the association with rivers, and identified possible new clues for an underlying cause.

Atypical Antibody Dynamics During Human Coronavirus HKU1 Infections.

Human coronavirus HKU1 (HCoV-HKU1) is one of the four endemic coronaviruses. It has been suggested that there is a difference in incidence, with PCR-confirmed HCoV-NL63 and HCoV-OC43 infections occurring more commonly, whereas HCoV-HKU1 is the least seen. Lower incidence of HCoV-HKU1 infection has also been observed in serological studies. The current study aimed to investigate antibody dynamics during PCR-confirmed HCoV-HKU1 infections using serum collected during infection and 1 month later. We expressed a new HCoV-HKU1 antigen consisting of both the linker and carboxy-terminal domain of the viral nucleocapsid protein and implemented it in ELISA. We also applied a spike-based Luminex assay on serum samples from PCR-confirmed infections by the four endemic HCoVs. At least half of HCoV-HKU1-infected subjects consistently showed no antibody rise via either assay, and some subjects even exhibited substantial antibody decline. Investigation of self-reported symptoms revealed that HCoV-HKU1-infected subjects rated their illness milder than subjects infected by other HCoVs. In conclusion, HCoV-HKU1 infections reported in this study displayed atypical antibody dynamics and milder symptoms when compared to the other endemic HCoVs.

Divergent Rhabdovirus Discovered in a Patient with New-Onset Nodding Syndrome.

A divergent rhabdovirus was discovered in the bloodstream of a 15-year-old girl with Nodding syndrome from Mundri West County in South Sudan. Nodding syndrome is a progressive degenerative neuropathy of unknown cause affecting thousands of individuals in Sub-Saharan Africa. The index case was previously healthy until she developed head-nodding seizures four months prior to presentation. Virus discovery by VIDISCA-NGS on the patient's plasma detected multiple sequence reads belonging to a divergent rhabdovirus. The viral load was 3.85 × 103 copies/mL in the patient's plasma and undetectable in her cerebrospinal fluid. Further genome walking allowed for the characterization of full coding sequences of all the viral proteins (N, P, M, U1, U2, G, U3, and L). We tentatively named the virus "Mundri virus" (MUNV) and classified it as a novel virus species based on the high divergence from other known viruses (all proteins had less than 43% amino acid identity). Phylogenetic analysis revealed that MUNV forms a monophyletic clade with several human-infecting tibroviruses prevalent in Central Africa. A bioinformatic machine-learning algorithm predicted MUNV to be an arbovirus (bagged prediction strength (BPS) of 0.9) transmitted by midges (BPS 0.4) with an artiodactyl host reservoir (BPS 0.9). An association between MUNV infection and Nodding syndrome was evaluated in a case-control study of 72 patients with Nodding syndrome (including the index case) matched to 65 healthy households and 48 community controls. No subject, besides the index case, was positive for MUNV RNA in their plasma. A serological assay detecting MUNV anti-nucleocapsid found, respectively, in 28%, 22%, and 16% of cases, household controls and community controls to be seropositive with no significant differences between cases and either control group. This suggests that MUNV commonly infects children in South Sudan yet may not be causally associated with Nodding syndrome.

No Evidence of Ntwetwe Virus Infections in Children Presenting to Kiboga Hospital, Uganda.

We investigated whether Ntwetwe virus-a novel orthobunyavirus discovered in a Ugandan girl with a fatal encephalopathy-was a common reason for hospital admission for children to Kiboga hospital, Uganda. A case-control was conducted between September 2019 and September 2020, including cases with severe neurological disease and mild febrile illness, matched to a healthy control without fever. Among 143 subjects, no cases with an acute infection were identified. This result suggests that Ntwetwe virus does not cause a major burden of disease amongst children presenting to Kiboga hospital during the study period.

Seasonal coronavirus protective immunity is short-lasting.

A key unsolved question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of acquired immunity. Insights from infections with the four seasonal human coronaviruses might reveal common characteristics applicable to all human coronaviruses. We monitored healthy individuals for more than 35 years and determined that reinfection with the same seasonal coronavirus occurred frequently at 12 months after infection.

Viral Metagenomics on Cerebrospinal Fluid.

Identifying the causative pathogen in central nervous system (CNS) infections is crucial for patient management and prognosis. Many viruses can cause CNS infections, yet screening for each individually is costly and time-consuming. Most metagenomic assays can theoretically detect all pathogens, but often fail to detect viruses because of their small genome and low viral load. Viral metagenomics overcomes this by enrichment of the viral genomic content in a sample. VIDISCA-NGS is one of the available workflows for viral metagenomics, which requires only a small input volume and allows multiplexing of multiple samples per run. The performance of VIDISCA-NGS was tested on 45 cerebrospinal fluid (CSF) samples from patients with suspected CNS infections in which a virus was identified and quantified by polymerase chain reaction. Eighteen were positive for an RNA virus, and 34 for a herpesvirus. VIDISCA-NGS detected all RNA viruses with a viral load >2 × 104 RNA copies/mL (n = 6) and 8 of 12 of the remaining low load samples. Only one herpesvirus was identified by VIDISCA-NGS, however, when withholding a DNase treatment, 11 of 18 samples with a herpesvirus load >104 DNA copies/mL were detected. Our results indicate that VIDISCA-NGS has the capacity to detect low load RNA viruses in CSF. Herpesvirus DNA in clinical samples is probably non-encapsidated and therefore difficult to detect by VIDISCA-NGS.

Novel Orthobunyavirus Identified in the Cerebrospinal Fluid of a Ugandan Child With Severe Encephalopathy.

A Ugandan child with an unexplained encephalitis was investigated using viral metagenomics. Several sequences from all segments of a novel orthobunyavirus were found. The S-segment, used for typing, showed 41% amino acid diversity to its closest relative. The virus was named Ntwetwe virus, after the hometown of the patient.