Jerome Bouquet

Erratum to: Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing

Author details Department of Laboratory Medicine, University of California, 185 Berry Street, San Francisco 94107 CA, USA. UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco 91407 CA, USA. Children’s Hospital Colorado and University of Colorado School of Medicine, Aurora, CO, USA. California Department of Public Health, Richmond, CA, USA. Kaiser Permanente Hospital, Oakland, CA, USA. John Muir Hospital, Walnut Creek, CA, USA. Department of Medicine, Division of Infectious Diseases, University of California, San Francisco 94107 CA, USA.

Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease

ABSTRACT Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. IMPORTANCE Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies. Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies.

RNA-Seq Analysis of Gene Expression, Viral Pathogen, and B-Cell/T-Cell Receptor Signatures in Complex Chronic Disease

Summary No differences in blood transcriptome, virome, and B-cell/T-cell receptor patterns were found between chronic fatigue syndrome or alternatively diagnosed chronic Lyme syndrome patients and controls. Neither disease was found to be associated with transcriptionally mediated immune dysfunction or active viral infection.

Genomic Epidemiology Reconstructs the Introduction and Spread of Zika Virus in Central America and Mexico

Summary The Zika virus (ZIKV) epidemic in the Americas established ZIKV as a major public health threat and uncovered its association with severe diseases, including microcephaly. However, genetic epidemiology in some at-risk regions, particularly Central America and Mexico, remains limited. We report 61 ZIKV genomes from this region, generated using metagenomic sequencing with ZIKV-specific enrichment, and combine phylogenetic, epidemiological, and environmental data to reconstruct ZIKV transmission. These analyses revealed multiple independent ZIKV introductions to Central America and Mexico. One introduction, likely from Brazil via Honduras, led to most infections and the undetected spread of ZIKV through the region from late 2014. Multiple lines of evidence indicate biannual peaks of ZIKV transmission in the region, likely driven by varying local environmental conditions for mosquito vectors and herd immunity. The spatial and temporal heterogeneity of ZIKV transmission in Central America and Mexico challenges arbovirus surveillance and disease control measures.

Metagenomic-based Surveillance of Pacific Coast tick Dermacentor occidentalis Identifies Two Novel Bunyaviruses and an Emerging Human Ricksettsial Pathogen

An increasing number of emerging tick-borne diseases has been reported in the United States since the 1970s. Using metagenomic next generation sequencing, we detected nucleic acid sequences from 2 novel viruses in the family Bunyaviridae and an emerging human rickettsial pathogen, Rickettsia philipii, in a population of the Pacific Coast tick, Dermacentor occidentalis in Mendocino County sampled annually from 2011 to 2014. A total of 250 adults of this human-biting, generalist tick were collected from contiguous chaparral and grassland habitats, and RNA from each individually extracted tick was deep sequenced to an average depth of 7.3 million reads. We detected a Francisella endosymbiont in 174 ticks (70%), and Rickettsia spp. in 19 ticks (8%); Rickettsia-infected ticks contained R. rhipicephali (16 of 250, 6.4%) or R. philipii (3 of 250,1.2%), the agent of eschar-associated febrile illness in humans. The genomes of 2 novel bunyaviruses (>99% complete) in the genera Nairovirus and Phlebovirus were also identified and found to be present in 20–91% of ticks, depending on the year of collection. The high prevalence of these bunyaviruses in sampled Dermacentor ticks suggests that they may be viral endosymbionts, although further studies are needed to determine whether they are infectious for vertebrate hosts, especially humans, and their potential role in tick ecology.

Differentiation enhances Zika virus infection of neuronal brain cells

Zika virus (ZIKV) is an emerging, mosquito-borne pathogen associated with a widespread 2015–2016 epidemic in the Western Hemisphere and a proven cause of microcephaly and other fetal brain defects in infants born to infected mothers. ZIKV infections have been also linked to other neurological illnesses in infected adults and children, including Guillain-Barré syndrome (GBS), acute flaccid paralysis (AFP) and meningoencephalitis, but the viral pathophysiology behind those conditions remains poorly understood. Here we investigated ZIKV infectivity in neuroblastoma SH-SY5Y cells, both undifferentiated and following differentiation with retinoic acid. We found that multiple ZIKV strains, representing both the prototype African and contemporary Asian epidemic lineages, were able to replicate in SH-SY5Y cells. Differentiation with resultant expression of mature neuron markers increased infectivity in these cells, and the extent of infectivity correlated with degree of differentiation. New viral particles in infected cells were visualized by electron microscopy and found to be primarily situated inside vesicles; overt damage to the Golgi apparatus was also observed. Enhanced ZIKV infectivity in a neural cell line following differentiation may contribute to viral neuropathogenesis in the developing or mature central nervous system.

Experimental Zika Virus Infection in a New World Monkey Model Reproduces Key Features of the Human Disease

Human infections by Zika virus (ZIKV), a mosquito-borne flavivirus, are associated with a current widespread outbreak in the Americas, and have been associated with neurological complications and adverse fetal outcomes such as microcephaly in pregnant women. A suitable non-human primate model is urgently needed. To evaluate ZIKV infectivity, pathogenesis, and persistence, we inoculated 4 marmosets with ZIKV and followed them by clinical monitoring and serial sampling of body fluids for up to 11 weeks. We found that marmosets experimentally infected with ZIKV reproduced key features of the human disease, including (1) asymptomatic infection, (2) brief period of detectable virus in serum (<1 week), (3) detection in other body fluids (urine, saliva, semen, and stool) for at least 2 weeks following acute infection, and (4) persistence in lymph nodes, but not other tissues, at 1 month post-infection. ZIKV-positive saliva and serum samples, but not urine, were found to be infectious in cell culture. By day 6 post-inoculation, most marmosets exhibited detectable neutralizing antibody responses concurrent with activation of NK cell and B cell subsets and an increase in circulating cytokines associated with type II interferon signaling, Transcriptome profiling revealed enrichment of immune responses to active viral infection, with up-regulation of both type I and II interferon signaling pathways, anduncovered potential host biomarkers. These results suggest that a New World monkey model of acute ZIKV infection mimics the human disease, and is likely to be useful for testing of drug and vaccine candidates.

Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the critical need for reference strain sequencing

Primary amoebic meningoencephalitis (PAM) is a rare, often lethal cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes. In this study, we present the first draft assembly of the Balamuthia mandrillaris genome recovered from a rare survivor of PAM, in total comprising 49 Mb of sequence. Comparative analysis of the mitochondrial genome and high-copy number genes from 6 additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, with the closest homologs corresponding to other amoebae, hydroids, algae, slime molds, and peat moss,. We also describe the use of unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year old girl. Real-time NGS testing of a hospital day 6 CSF sample detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better outcome, would have required availability of the complete genome sequence. These results underscore the diverse evolutionary origins underpinning this eukaryotic pathogen, and the critical importance of whole-genome reference sequences for microbial detection by NGS.