Bioinformatics resources for SARS-CoV-2 discovery and surveillance.

In early January 2020, the novel coronavirus (SARS-CoV-2) responsible for a pneumonia outbreak in Wuhan, China, was identified using next-generation sequencing (NGS) and readily available bioinformatics pipelines. In addition to virus discovery, these NGS technologies and bioinformatics resources are currently being employed for ongoing genomic surveillance of SARS-CoV-2 worldwide, tracking its spread, evolution and patterns of variation on a global scale. In this review, we summarize the bioinformatics resources used for the discovery and surveillance of SARS-CoV-2. We also discuss the advantages and disadvantages of these bioinformatics resources and highlight areas where additional technical developments are urgently needed. Solutions to these problems will be beneficial not only to the prevention and control of the current COVID-19 pandemic but also to infectious disease outbreaks of the future.

Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metatranscriptomic sequencing.

Encephalitis is most often caused by a variety of infectious agents identified through diagnostic tests utilizing cerebrospinal fluid. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic sequencing of residual clinical samples from multiple tissue types and independent clinical review. Forty-three specimens were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing ('metatranscriptomics') to determine the presence and abundance of potential pathogens, and to describe the possible aetiologies of unexplained encephalitis. Using this protocol, we identified five RNA and two DNA viruses associated with human infection from both non-sterile and sterile sites, which were confirmed by PCR. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases. Immune-mediated encephalitis was considered likely in six cases and metatranscriptomics did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasizes the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that non-central-nervous-system sampling in encephalitis clinical guidelines and protocols could improve the diagnostic yield.

Next Generation Sequencing and Bioinformatics Methodologies for Infectious Disease Research and Public Health: Approaches, Applications, and Considerations for Development of Laboratory Capacity.

Next generation sequencing (NGS) combined with bioinformatics has successfully been used in a vast array of analyses for infectious disease research of public health relevance. For instance, NGS and bioinformatics approaches have been used to identify outbreak origins, track transmissions, investigate epidemic dynamics, determine etiological agents of a disease, and discover novel human pathogens. However, implementation of high-quality NGS and bioinformatics in research and public health laboratories can be challenging. These challenges mainly include the choice of the sequencing platform and the sequencing approach, the choice of bioinformatics methodologies, access to the appropriate computation and information technology infrastructure, and recruiting and retaining personnel with the specialized skills and experience in this field. In this review, we summarize the most common NGS and bioinformatics workflows in the context of infectious disease genomic surveillance and pathogen discovery, and highlight the main challenges and considerations for setting up an NGS and bioinformatics-focused infectious disease research public health laboratory. We describe the most commonly used sequencing platforms and review their strengths and weaknesses. We review sequencing approaches that have been used for various pathogens and study questions, as well as the most common difficulties associated with these approaches that should be considered when implementing in a public health or research setting. In addition, we provide a review of some common bioinformatics tools and procedures used for pathogen discovery and genome assembly, along with the most common challenges and solutions. Finally, we summarize the bioinformatics of advanced viral, bacterial, and parasite pathogen characterization, including types of study questions that can be answered when utilizing NGS and bioinformatics.

Application of Pathogen Discovery/Metagenomic Sequencing in CNS HIV.

PURPOSE OF REVIEW: Neurological conditions associated with HIV/AIDS including central nervous system (CNS), opportunistic infections (OI), chronic conditions including HIV-associated neurocognitive disorder, and cerebrospinal fluid (CSF) viral escape remain major contributors to morbidity and mortality worldwide. CNS infections in HIV-infected patients are often challenging to diagnose by traditional microbiological testing, impacting treatment and outcome. RECENT FINDINGS: Recent advances in diagnostic techniques, including metagenomic next-generation sequencing (mNGS), are changing the landscape of microbiological testing, mainly in resource-rich settings. Pathogen discovery techniques offer a hypothesis-free approach to diagnostic testing, yielding comprehensive analysis of microbial genetic material. Given the extent of genetic material produced, deep sequencing tools not only hold promise in the diagnosis of CNS infections but also in defining key pathogenic steps which have previously been unanswered. Significant challenges remain to implementing pathogen discovery techniques in routine clinical practice including cost, expertise and infrastructure needed including laboratory and bioinformatics support, and sample contamination risk. The use in resource-limited regions where the burden of CNS complications due to HIV/AIDS is highest remains poorly defined. Though, major opportunities utilizing pathogen discovery techniques exist to enhance surveillance and diagnosis and improve our understanding of mechanisms of neuroinvasion in CNS conditions associated with HIV.

Metagenomic Next-Generation Sequencing of the 2014 Ebola Virus Disease Outbreak in the Democratic Republic of the Congo.

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.

Isolation of Complete Equine Encephalitis Virus Genome from Human Swab Specimen, Peru.

While studying respiratory infections in Peru, we identified Venezuelan equine encephalitis virus (VEEV) in a nasopharyngeal swab, indicating that this alphavirus can be present in human respiratory secretions. Because VEEV may be infectious when aerosolized, our finding is relevant for the management of VEEV-infected patients and for VEEV transmission studies.

Viral Surveillance in Serum Samples From Patients With Acute Liver Failure By Metagenomic Next-Generation Sequencing.

BACKGROUND: Twelve percent of all acute liver failure (ALF) cases are of unknown origin, often termed indeterminate. A previously unrecognized hepatotropic virus has been suspected as a potential etiologic agent. METHODS: We compared the performance of metagenomic next-generation sequencing (mNGS) with confirmatory nucleic acid testing (NAT) to routine clinical diagnostic testing in detection of known or novel viruses associated with ALF. Serum samples from 204 adult ALF patients collected from 1998 to 2010 as part of a nationwide registry were analyzed. One hundred eighty-seven patients (92%) were classified as indeterminate, while the remaining 17 patients (8%) served as controls, with infections by either hepatitis A virus or hepatitis B virus (HBV), or a noninfectious cause for their ALF. RESULTS: Eight cases of infection from previously unrecognized viral pathogens were detected by mNGS (4 cases of herpes simplex virus type 1, including 1 case of coinfection with HBV, and 1 case each of HBV, parvovirus B19, cytomegalovirus, and human herpesvirus 7). Several missed dual or triple infections were also identified, and assembled viral genomes provided additional information on genotyping and drug resistance mutations. Importantly, no sequences corresponding to novel viruses were detected. CONCLUSIONS: These results suggest that ALF patients should be screened for the presence of uncommon viruses and coinfections, and that most cases of indeterminate ALF in the United States do not appear to be caused by novel viral pathogens. In the future, mNGS testing may be useful for comprehensive diagnosis of viruses associated with ALF, or to exclude infectious etiologies.

Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients.

BACKGROUND: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. METHODS: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. RESULTS: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. CONCLUSIONS: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing.

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.

Pathogen Discovery in the Post-COVID Era.

Pathogen discovery plays a crucial role in the fields of infectious diseases, clinical microbiology, and public health. During the past four years, the global response to the COVID-19 pandemic highlighted the importance of early and accurate identification of novel pathogens for effective management and prevention of outbreaks. The post-COVID era has ushered in a new phase of infectious disease research, marked by accelerated advancements in pathogen discovery. This review encapsulates the recent innovations and paradigm shifts that have reshaped the landscape of pathogen discovery in response to the COVID-19 pandemic. Primarily, we summarize the latest technology innovations, applications, and causation proving strategies that enable rapid and accurate pathogen discovery for both acute and historical infections. We also explored the significance and the latest trends and approaches being employed for effective implementation of pathogen discovery from various clinical and environmental samples. Furthermore, we emphasize the collaborative nature of the pandemic response, which has led to the establishment of global networks for pathogen discovery.

Broad range molecular detection methods identify only Borrelia spp. in erythema migrans biopsies and blood of tick-bitten patients.

In this multicenter study conducted in France, we challenged the hypothesis of the transmission of pathogens other than Borrelia spp. in 22 patients developing erythema migrans following a tick bite. Using a combination of high-throughput microfluidic PCRs and agnostic metagenomics on skin biopsies and blood samples, no microorganisms other than Borrelia spp. was found.

Novel Bartonella agent as cause of verruga peruana.

While studying chronic verruga peruana infections in Peru from 2003, we isolated a novel Bartonella agent, which we propose be named Candidatus Bartonella ancashi. This case reveals the inherent weakness of relying solely on clinical syndromes for diagnosis and underscores the need for a new diagnostic paradigm in developing settings.

Metagenomics analysis reveals presence of the Merida-like virus in Georgia.

Arbovirus surveillance is fundamental for the discovery of novel viruses and prevention of febrile vector-borne illnesses. Vector-borne pathogens can rapidly expand and adapt in new geographic and environmental conditions. In this study, metagenomic surveillance was conducted to identify novel viruses in the Country of Georgia. A total of 521 mosquitoes were captured near a military training facility and pooled from species Culex pipiens (Linnaeus) (87%) and Aedes albopictus (Skuse) (13%). We decided to further analyze the Culex pipiens mosquitoes, due to the more extensive number of samples collected. Our approach was to utilize an unbiased total RNA-seq for pathogen discovery in order to explore the mosquito virome. The viral reads from this analysis were mostly aligned to Insect-specific viruses from two main families, the Iflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a negative- and single-stranded RNA virus. Our pathogen discovery analysis revealed viral reads aligning to the Merida-like virus Turkey (MERDLVT) strain among the Rhabdoviridae. To further validate this result, we conducted a BLAST sequence comparison analysis of our samples with the MERDLVT strain. Our positive samples aligned to the MERDLVT strain with 96-100% sequence identity and 99.7-100% sequence coverage. A bootstrapped maximum-likelihood phylogenetic tree was used to evaluate the evolutionary relationships among these positive pooled specimens with the (MERDLVT) strain. The Georgia samples clustered most closely with two strains from Turkey, the Merida-like virus KE-2017a isolate 139-1-21 and the Merida-like virus Turkey isolate P431. Collectively, these results show the presence of the MERDLVT strain in Georgia.

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

Case report: nanopore targeted sequencing in the diagnosis of invasive pulmonary Aspergillus infection in a patient with acute promyelocytic leukemia.

Invasive pulmonary aspergillosis (IPA) is an infectious disease with a high mortality rate due to diagnostic difficulties associated with the lack of a typical clinical presentation and the inadequacy of the available laboratory testing methods. Nanopore targeted sequencing (NTS) is an alternative method of broad-based pathogen discovery, associated with rapid turnaround and high accuracy. This case report presents a patient with IPA and acute promyelocytic leukemia, diagnosed using the NTS method, which detected Aspergillus flavus in the patient's blood and pleural fluid. The patient was treated effectively with antifungal therapy. Early diagnosis of IPA improved long-term patient prognosis and quality of life.

Building a global immune system.

The COVID-19 pandemic exposed global vulnerabilities to emerging infectious diseases, heralded by earlier outbreaks, that did not result in appropriate investments in surveillance, international collaboration, and response. We propose specific steps that should be taken to reduce future risks to public health, economic and political stability, and food security.

Discovery of Rickettsia spp. in mosquitoes collected in Georgia by metagenomics analysis and molecular characterization.

Arthropods have a broad and expanding worldwide presence and can transmit a variety of viral, bacterial, and parasite pathogens. A number of Rickettsia and Orientia species associated with ticks, fleas, lice, and mites have been detected in, or isolated from, patients with febrile illness and/or animal reservoirs throughout the world. Mosquitoes are not currently considered vectors for Rickettsia spp. pathogens to humans or to animals. In this study, we conducted a random metagenome next-generation sequencing (NGS) of 475 pools of Aedes, Culex, and Culiseta species of mosquitoes collected in Georgia from 2018 to 2019, identifying rickettsial gene sequences in 33 pools of mosquitoes. We further confirmed the findings of the Rickettsia by genus-specific quantitative PCR (qPCR) and multi-locus sequence typing (MLST). The NGS and MLST results indicate that Rickettsia spp. are closely related to Rickettsia bellii, which is not known to be pathogenic in humans. The results, together with other reports of Rickettsia spp. in mosquitoes and the susceptibility and transmissibility experiments, suggest that mosquitoes may play a role in the transmission cycle of Rickettsia spp.

Culture-Independent Discovery of Viroids by Deep Sequencing and Computational Algorithms.

Viroids are single-stranded circular RNA molecules that cause diseases in plants and do not encode any protein. Classical approaches for the identification of new viroids are challenging for many plant pathology laboratories as viroid cDNA synthesis and sequencing require purification and enrichment of the naked viroid RNA by two-dimensional gel electrophoresis. Conventional metagenomic approaches are not effective for viroid discovery because the total number of known viroids is small, and distinct viroids share limited nucleotide sequence similarity. In this chapter, we describe a homology-independent approach for the identification of both known and new viroids in disease samples. It is known that viroid infection of plants triggers production of overlapping viroid-derived small interfering RNAs (siRNAs) targeting the entire genome with high densities and that replication of viroids occurs via a rolling-circle mechanism to yield head-to-tail multiple-repeat replicative intermediates. Our approach involves deep sequencing of either long or small RNAs in a disease sample followed by viroid identification with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Among the sequenced total small RNAs, PFOR retains viroid-derived siRNAs for viroid genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, a unique feature of viroid RNA replication. In contrast, long RNAs sequenced after depletion of ribosomal RNAs are cut into 21-nucleotide virtual overlapping small RNAs with the algorithm SLS (splitting longer read into shorter fragments) before PFOR. We show that new viroids or viroids from the two known families are readily identified and their full-length sequences recovered by PFOR from long or small RNAs sequenced directly from infected plants. We propose that our approach can be used for viroid discovery in both plants and potentially animals since PFOR identifies viroids by searching for circular RNAs or a unique replication intermediate of the viroid genome in a sequence homology-independent manner.

Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider.

Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human-animal interfaces in geographical hotspots for emerging infectious diseases. Where human-animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics.

Discovery and Surveillance of Tick-Borne Pathogens.

Within the past 30 yr molecular assays have largely supplanted classical methods for detection of tick-borne agents. Enhancements provided by molecular assays, including speed, throughput, sensitivity, and specificity, have resulted in a rapid increase in the number of newly characterized tick-borne agents. The use of unbiased high throughput sequencing has enabled the prompt identification of new pathogens and the examination of tick microbiomes. These efforts have led to the identification of hundreds of new tick-borne agents in the last decade alone. However, little is currently known about the majority of these agents beyond their phylogenetic classification. Our article outlines the primary methods involved in tick-borne agent discovery and the current status of our understanding of tick-borne agent diversity.