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.

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.

Background: Metagenomic sequencing has the potential to identify a wide range of pathogens in human tissue samples. Sarcoidosis is a complex disorder whose etiology remains unknown and for which a variety of infectious causes have been hypothesized. We sought to conduct metagenomic sequencing on cases of ocular and periocular sarcoidosis, none of them with previously identified infectious causes. Methods: Archival tissue specimens of 16 subjects with biopsies of ocular and periocular tissues that were positive for non-caseating granulomas were used as cases. Four archival tissue specimens that did not demonstrate non-caseating granulomas were also included as controls. Genomic DNA was extracted from tissue sections. DNA libraries were generated from the extracted genomic DNA and the libraries underwent next-generation sequencing. Results: We generated between 4.8 and 20.7 million reads for each of the 16 cases plus four control samples. For eight of the cases, we identified microbial pathogens that were present well above the background, with one potential pathogen identified for seven of the cases and two possible pathogens for one of the cases. Five of the eight cases were associated with bacteria ( Campylobacter concisus, Neisseria elongata, Streptococcus salivarius, Pseudopropionibacterium propionicum, and Paracoccus yeei), two cases with fungi ( Exophiala oligosperma, Lomentospora prolificans and Aspergillus versicolor) and one case with a virus (Mupapillomavirus 1). Interestingly, four of the five bacterial species are also part of the human oral microbiome. Conclusions: Using a metagenomic sequencing we identified possible infectious causes in half of the ocular and periocular sarcoidosis cases analyzed. Our findings support the proposition that sarcoidosis could be an etiologically heterogenous disease. Because these are previously banked samples, direct follow-up in the respective patients is impossible, but these results suggest that sequencing may be a valuable tool in better understanding the etiopathogenesis of sarcoidosis and in diagnosing and treating this disease.

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.

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.

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.

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.

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.

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.

The seal picornavirus 1, species Aquamavirus A, is currently the only recognized member of the genus Aquamavirus within the family Picornaviridae. The bear picornavirus 1 was recently proposed as the second species in the genus under the name aquamavirus B. Herein, we determined the complete genomes of two novel pinniped picornaviruses, the harbor seal picornavirus (HsPV) and the ribbon seal picornavirus (RsPV). The HsPV and the RsPV were isolated in Vero.DogSLAMtag cells from samples collected from stranded harbor (Phoca vitulina) and ribbon (Histriophoca fasciata) seals. RsPV-infected Vero.DogSLAMtag cells displaying extensive cytopathic effects were processed for transmission electron microscopy and revealed non-enveloped viral particles aggregated into paracrystalline arrays in the cytoplasm. A next-generation sequencing approach was used to recover the complete genomes of the HsPV and the RsPV (6,709 and 6,683 bp, respectively). Phylogenetic and genetic analyses supported the HsPV and the RsPV as members of the Aquamavirus genus. Based on these results, RsPV represents a novel strain of Aquamavirus A, while the HsPV is a novel strain of the proposed species aquamavirus B. These discoveries provide information on the evolutionary relationships and ultrastructure of aquamaviruses and expands the known host range of those viruses. Our results underscore the importance of the application of classical virology and pathology techniques coupled with high-throughput sequencing technologies for the discovery and characterization of pathogens in wild marine mammals.