Genomic and serologic characterization of enterovirus A71 brainstem encephalitis

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.Grants supporting this project include the National Multiple Sclerosis Society and the American Academy of Neurology award FAN-1608-25607 (R.D.S.), Clinical Research Training Scholarship P0534134 (P.S.R.), Sandler and William K. Bowes Jr Foundations (M.R.W., J.L.D., L.M.K., H.A.S., K.C.Z.), Rachleff Family Foundation (M.R.W.), and NINDS of the NIH under award K08NS096117 (M.R.W.) and F31NS113432 (K.E.L.). This study was partially supported by a grant from the Spanish National Health Institute [grant number PI15CIII-00020] and the European Regional Development Fund (FEDER funds). UCSF Biomedical Sciences Program (I.A.H., K.E.L.), UCSF Medical Scientist Training Program (K.E.L.), and the Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., J.L.D., E.D.C.) also supported this project.S

The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein

Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1). Endogenous UPF1 interacted with the ZIKV capsid protein in coimmunoprecipitation experiments, and capsid expression posttranscriptionally downregulated UPF1 protein levels, a process that we confirmed occurs during ZIKV infection. Cellular fractionation studies show that the ZIKV capsid protein specifically targets nuclear UPF1 for degradation via the proteasome. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures, consistent with a model in which NMD restricts ZIKV infection in the fetal brain. We propose that ZIKV, via the capsid protein, has evolved a strategy to lower UPF1 levels and dampen antiviral activities of NMD, which in turn contributes to neuropathology in vivo

Fatal Powassan Encephalitis (Deer Tick Virus, Lineage II) in a Patient With Fever and Orchitis Receiving Rituximab.

Importance:Powassan virus is a rare but increasingly recognized cause of severe neurological disease. Objective:To highlight the diagnostic challenges and neuropathological findings in a fatal case of Powassan encephalitis caused by deer tick virus (lineage II) in a patient with follicular lymphoma receiving rituximab, with nonspecific anti-GAD65 antibodies, who was initially seen with fever and orchiepididymitis. Design, Setting, and Participants:Comparison of clinical, radiological, histological, and laboratory findings, including immunohistochemistry, real-time polymerase chain reaction, antibody detection, and unbiased sequencing assays, in a single case report (first seen in December 2016) at an academic medical center. Exposure:Infection with Powassan virus. Main Outcomes and Measures:Results of individual assays compared retrospectively. Results:In a 63-year-old man with fatal Powassan encephalitis, serum and cerebrospinal fluid IgM antibodies were not detected via standard methods, likely because of rituximab exposure. Neuropathological findings were extensive, including diffuse leptomeningeal and parenchymal lymphohistiocytic infiltration, microglial proliferation, marked neuronal loss, and white matter microinfarctions most severely involving the cerebellum, thalamus, and basal ganglia. Diagnosis was made after death by 3 independent methods, including demonstration of Powassan virus antigen in brain biopsy and autopsy tissue, detection of viral RNA in serum and cerebrospinal fluid by targeted real-time polymerase chain reaction, and detection of viral RNA in cerebrospinal fluid by unbiased sequencing. Extensive testing for other etiologies yielded negative results, including mumps virus owing to prodromal orchiepididymitis. Low-titer anti-GAD65 antibodies identified in serum, suggestive of limbic encephalitis, were not detected in cerebrospinal fluid. Conclusions and Relevance:Owing to the rarity of Powassan encephalitis, a high degree of suspicion is required to make the diagnosis, particularly in an immunocompromised patient, in whom antibody-based assays may be falsely negative. Unbiased sequencing assays have the potential to detect uncommon infectious agents and may prove useful in similar scenarios

Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19

Abstract The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov–Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19

Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19.

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19

Central Nervous System Virus Infection in African Children with Cerebral Malaria.

We aimed to identify the contribution of central nervous system (CNS) viral coinfection to illness in African children with retinopathy-negative or retinopathy-positive cerebral malaria (CM). We collected cerebrospinal fluid (CSF) from 272 children with retinopathy-negative or retinopathy-positive CM and selected CSF from 111 of these children (38 retinopathy positive, 71 retinopathy negative, 2 retinopathy unknown) for analysis by metagenomic next-generation sequencing. We found CSF viral coinfections in 7/38 (18.4%) retinopathy-positive children and in 18/71 (25.4%) retinopathy-negative children. Excluding HIV-1, human herpesviruses (HHV) represented 61% of viruses identified. Excluding HIV-1, CNS viral coinfection was equally likely in children who were retinopathy positive and retinopathy negative (P = 0.1431). Neither mortality nor neurological morbidity was associated with the presence of virus (odds ratio [OR] = 0.276, 95% CI: 0.056–1.363). Retinopathy-negative children with a higher temperature, lower white blood cell count, or being dehydrated were more likely to have viral coinfection. Level of consciousness at admission was not associated with CNS viral coinfection in retinopathy-negative children. Viral CNS coinfection is unlikely to contribute to coma in children with CM. The herpesviruses other than herpes simplex virus may represent incidental bystanders in CM, reactivating during acute malaria infection

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis.

In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF