Emerging Coronavirus Infections Research Articles

Від епідемій минулого до сьогодення і хвороби х, досвід боротьби та перспективи

People have faced epidemics and pandemics since ancient times, without yet understanding either their nature, their consequences, or how to protect themselves and prevent their spread. Each infectious disease has its own history, features related to the virulence of the pathogen, the mechanism of its transmission, contagiousness, which depend on the epidemic potential of the pathogen, the intensity of the epidemic process of infection, and the consequences for eachindividual and society in general. Every year, new human microorganisms appear, among which viruses play a leading role,new parasitic systems are formed or already existing parasitic systems change, in which a person becomes the biological host of one or another pathogen. It is shown that zoonotic microorganisms prevail among human pathogens, in particular among emergent ones. Viruses predominate among pathogens with epidemic and pandemic potential. The article examines the chronology of major epidemics and pandemics, starting with an unknown disease in China 5,000 years ago and ending with monkeypox, the spread of which began in May 2022. Considerable attention is paid to current infectious diseases with pandemic potential, namely zoonotic influenza, emerging coronavirus infections (SARS, MERS, COVID-19), Ebola and Zika diseases, re-emerging monkeypox. A list of 10 potentially pandemic infectious diseases, which WHO has identified as priority, is provided, which also includes disease X. Taking into account the lessons of the COVID-19 pandemic, all countries, including Ukraine, need to make a lot more efforts to achieve a truly adequate level of preparedness for preventing biological threats and responding to future challenges. It is noted that preparations are currently underway for the adoption of the WHO Convention, an agreement or another international document on pandemic prevention, preparedness and response, the draft of which is currently undergoing the coordination stage. The implementation of its provisions at the international and national levels will contribute to increasing the effectiveness of international and national biosecurity systems and strengthening the potential for combating biological threats. It is emphasized that the national biosafety system in Ukraine needs significant improvement in the direction of strengthening the system of epidemiological surveillance of infectious diseases. Key words: epidemic, pandemic, emergent infection, pathogen, MERS, COVID-19, monkeypox, disease X.

Investigation of the Host Kinome Response to Coronavirus Infection Reveals PI3K/mTOR Inhibitors as Betacoronavirus Antivirals.

Host kinases play essential roles in the host cell cycle, innate immune signaling, the stress response to viral infection, and inflammation. Previous work has demonstrated that coronaviruses specifically target kinase cascades to subvert host cell responses to infection and rely upon host kinase activity to phosphorylate viral proteins to enhance replication. Given the number of kinase inhibitors that are already FDA approved to treat cancers, fibrosis, and other human disease, they represent an attractive class of compounds to repurpose for host-targeted therapies against emerging coronavirus infections. To further understand the host kinome response to betacoronavirus infection, we employed multiplex inhibitory bead mass spectrometry (MIB-MS) following MERS-CoV and SARS-CoV-2 infection of human lung epithelial cell lines. Our MIB-MS analyses revealed activation of mTOR and MAPK signaling following MERS-CoV and SARS-CoV-2 infection, respectively. SARS-CoV-2 host kinome responses were further characterized using paired phosphoproteomics, which identified activation of MAPK, PI3K, and mTOR signaling. Through chemogenomic screening, we found that clinically relevant PI3K/mTOR inhibitors were able to inhibit coronavirus replication at nanomolar concentrations similar to direct-acting antivirals. This study lays the groundwork for identifying broad-acting, host-targeted therapies to reduce betacoronavirus replication that can be rapidly repurposed during future outbreaks and epidemics. The proteomics, phosphoproteomics, and MIB-MS datasets generated in this study are available in the Proteomics Identification Database (PRIDE) repository under project identifiers PXD040897 and PXD040901.

Novel Molecular Consortia of Cannabidiol with Nonsteroidal Anti-Inflammatory Drugs Inhibit Emerging Coronaviruses' Entry.

The COVID-19 pandemic provoked a global health crisis and highlighted the need for new therapeutic strategies. In this study, we explore the potential of the molecular consortia of cannabidiol (CBD) and non-steroidal anti-inflammatory drugs (NSAIDs) as novel antiviral dual-target agents against SARS-CoV-2/COVID-19. CBD is a natural compound with a wide range of therapeutic activities, including antiviral and anti-inflammatory properties, while NSAIDs are commonly used to mitigate the symptoms of viral infections. Chemical modifications of CBD with NSAIDs were performed to obtain dual-target agents with enhanced activity against SARS-CoV-2. The synthesised compounds were characterised using spectroscopic techniques. The biological activity of three molecular consortia (CBD-ibuprofen, CBD-ketoprofen, and CBD-naproxen) was evaluated in cell lines transduced with vesicular stomatitis virus-based pseudotypes bearing the SARS-CoV-1 or SARS-CoV-2 spike proteins or infected with influenza virus A/Puerto Rico/8/34. The results showed that some CBD-NSAID molecular consortia have superior antiviral activity against SARS-CoV-1 and SARS-CoV-2, but not against the influenza A virus. This may suggest a potential therapeutic role for these compounds in the treatment of emerging coronavirus infections. Further studies are needed to investigate the efficacy of these compounds in vivo, and their potential use in clinical settings. Our findings provide a promising new approach to combatting current and future viral emergencies.

Pantropic canine coronavirus induces canine M1 macrophage polarization in vitro

Emerging coronavirus infections are a major threat to global public health. In this respect, a novel recombination of canine coronavirus (CCoV) and feline coronavirus (FCoV) was described among human biological samples, giving rise to a potential zoonosis. Despite all efforts, the host‒virus immune response related to CCoV is still unknown. In this study, pantropic CCoV infection of canine macrophages, derived from peripheral blood monocytes, was performed. After infection, macrophages were first polarized to the M1 and/or M2 phenotype. Moreover, infection kinetics, cell viability, apoptosis, mitochondrial dysfunction associated with reactive oxygen species and oxide nitric production were measured. Our results demonstrated that virus infection mainly polarized host macrophages to the classically activated (M1) phenotype, as demonstrated by amoeboid morphology with numerous fibrillary cytoplasmic processes followed by classical phenotypes. Viral infection released new particles 18 h post-infection associated with a decrease in viable cells. Furthermore, upon CCoV infection, M1 cells exhibited reduced phagocytosis properties, as evidenced by a neutral red uptake assay. This in vitro method opens an avenue for further studies on host-virus interaction.

How COVID-19 exposed pre-existing roadblocks for cancer control in Africa: strategies, lessons and recommendations from the 2019-2020 Africa Cancer Research and Control ECHO.

The COVID-19 related mitigation measures adversely affected various cancer control activities in Africa, with cancer prevention and screening activities amongst the most significantly impacted. When the COVID-19 pandemic struck, the Africa Cancer Research and Control ECHO utilised their virtual platform to share experiences and knowledge of how to continue cancer service delivery during the pandemic. This analysis describes the evolved strategies, dilemmas, and recommendations to strengthen the health systems for cancer control in Africa. Eleven 1-hour-long sessions about the then newly emerging coronavirus infection and its impact on cancer control in Africa were held from April 2020 to August 2020, using Zoom®. An average of 39 participants attended the sessions including scientists, clinicians, policymakers and global partners. Sessions were analysed thematically. Most strategies to maintain cancer services during the COVID-19 pandemic centred around cancer treatment, with few strategies on maintaining cancer prevention services, early detection, palliative care and research services. The most mentioned challenge during the pandemic was fear of exposure to COVID-19 infection at the health facility during diagnosis, treatment or follow-up for cancer care. Other challenges were disruptions to service delivery, inaccessibility of cancer treatment, disruption of research activities and a lack of psychosocial support for COVID-19 related fear/anxiety. Significantly, this analysis shows that the COVID-19 related mitigation measures exacerbated existing predicaments in Africa, such as inadequate attention to cancer prevention strategies, psychosocial and palliative services and cancer research. The Africa Cancer ECHO recommends African countries to leverage the infrastructure developed in response to COVID-19 pandemic to strengthen the health system along the entire cancer control continuum. This calls for urgent action to develop and implement evidence-based frameworks and comprehensive National Cancer Control Plans that will withstand any future disruptions.

Anticoronaviral Activity of the Natural Phloroglucinols, Dryocrassin ABBA and Filixic Acid ABA from the Rhizome of Dryopteris crassirhizoma by Targeting the Main Protease of SARS-CoV-2.

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5–12.6 h) and high plasma exposure (AUC 19.3–65 μg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

An Update on Animal Models for Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Countermeasure Development.

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic since March 2020 and led to significant challenges to over 200 countries and regions all over the world. The establishment of highly pathogenic coronavirus animal model is beneficial for the study of vaccines and pathogenic mechanism of the virus. Laboratory mice, Syrian hamsters, Non-human primates and Ferrets have been used to establish animal models of emerging coronavirus infection. Different animal models can reproduce clinical infection symptoms at different levels. Appropriate animal models are of great significance for the pathogenesis of COVID-19 and the research progress related to vaccines. This review aims to introduce the current progress about experimental animal models for SARS-CoV-2, and collectively generalize critical aspects of disease manifestation in humans and increase their usefulness in research into COVID-19 pathogenesis and developing new preventions and treatments.

Broad Spectrum Antiviral Properties of Cardiotonic Steroids Used as Potential Therapeutics for Emerging Coronavirus Infections.

Cardiotonic steroids are steroid-like natural compounds known to inhibit Na+/K+-ATPase pumps. To develop a broad-spectrum antiviral drug against the emerging coronavirus infection, this study assessed the antiviral properties of these compounds. The activity of seven types of cardiotonic steroids against the MERS-CoV, SARS-CoV, and SARS-CoV-2 coronavirus varieties was analyzed using immunofluorescence antiviral assay in virus-infected cells. Bufalin, cinobufagin, and telocinobufagin showed high anti-MERS-CoV activities (IC50, 0.017~0.027 μM); bufalin showed the most potent anti-SARS-CoV and SARS-CoV-2 activity (IC50, 0.016~0.019 μM); cinobufotalin and resibufogenin showed comparatively low anti-coronavirus activity (IC50, 0.231~1.612 μM). Differentially expressed genes in Calu3 cells treated with cinobufagin, telocinobufagin, or bufalin, which had high antiviral activity during MERS-CoV infection were analyzed using QuantSeq 3′ mRNA-Seq analysis and data showed similar gene expression patterns. Furthermore, the intraperitoneal administration of 10 mg/kg/day bufalin, cinobufagin, or digitoxin induced 100% death after 1, 2, and 4 days in 5-day repeated dose toxicity studies and it indicated that bufalin had the strongest toxicity. Pharmacokinetic studies suggested that telocinobufagin, which had high anti-coronavirus activity and low toxicity, had better microsomal stability, lower CYP inhibition, and better oral bioavailability than cinobufagin. Therefore, telocinobufagin might be the most promising cardiotonic steroid as a therapeutic for emerging coronavirus infections, including COVID-19.

The Potential of Medicinal Plants and Bioactive Compounds in the Fight Against COVID-19

The Potential of Medicinal Plants and Bioactive Compounds in the Fight Against COVID-19

Potential for Developing Plant-Derived Candidate Vaccines and Biologics against Emerging Coronavirus Infections.

The emerging human coronavirus infections in the 21st century remain a major public health crisis causing worldwide impact and challenging the global health care system. The virus is circulating in several zoonotic hosts and continuously evolving, causing occasional outbreaks due to spill-over events occurring between animals and humans. Hence, the development of effective vaccines or therapeutic interventions is the current global priority in order to reduce disease severity, frequent outbreaks, and to prevent future infections. Vaccine development for newly emerging pathogens takes a long time, which hinders rapid immunization programs. The concept of plant-based pharmaceuticals can be readily applied to meet the recombinant protein demand by means of transient expression. Plants are evolved as an expression platform, and they bring a combination of unique interests in terms of rapid scalability, flexibility, and economy for industrial-scale production of effective vaccines, diagnostic reagents, and other biopharmaceuticals. Plants offer safe biologics to fulfill emergency demands, especially during pandemic situations or outbreaks caused by emerging strains. This review highlights the features of a plant expression platform for producing recombinant biopharmaceuticals to combat coronavirus infections with emphasis on COVID-19 vaccine and biologics development.

SARS-CoV-2 RdRp Inhibitors Selected from a Cell-Based SARS-CoV-2 RdRp Activity Assay System.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), urgently needs effective prophylactic and therapeutic drugs. RNA-dependent RNA polymerase (RdRp), essential for replicating and transcribing a viral RNA genome, is highly conserved in coronaviruses; thus, it is a potential target for inhibiting coronavirus infection. In this study, we generated the cell-based SARS-CoV-2 RdRp activity assay system by modifying a previously reported cell-based MERS-CoV RdRp activity assay system to screen for SARS-CoV-2 RdRp inhibitors. The assay system consisted of an expression plasmid encoding SARS-CoV-2 RdRp and an RdRp activity reporter plasmid. RdRp activity in the cells could be conveniently detected by luminescence after transfection. We confirmed that SARS-CoV-2 RdRp replicated double-stranded RNA using immunofluorescence staining and the inhibition of RdRp activity by remdesivir and lycorine using this system. Moreover, the Z-factor of this system was calculated to be 0.798, suggesting the reproducibility and reliability of the high-throughput screening system. Finally, we screened nucleoside and nucleotide analogs and identified adefovir dipivoxil, emtricitabine, telbivudine, entecavir hydrate, moroxydine and rifampin as novel SARS-CoV-2 RdRp inhibitors and therapeutic candidates for COVID-19 This system provides an effective high-throughput screening system platform for developing potential prophylactic and therapeutic drugs for COVID-19 and emerging coronavirus infections.

Overview of an Emerging Coronavirus Infection, COVID-19: Current Status of Vaccine Development and Therapeutics

Coronavirus infectious disease 2019 (COVID-19) first reported in Wuhan, China, causes serious respiratory illnesses such as lung failure and pneumonia. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) the pathogenic agent of COVID-19 has been confirmed as a novel coronavirus. WHO announced COVID-19 a global pandemic and now the whole world is eagerly waiting for vaccines and therapeutic treatment to get rid of this unstoppable coronavirus. As COVID-19 infection, a global threat creates unwanted human casualties and serious economic loss. To stop the ongoing uncontrolled situation researchers are racing to develop prevention and treatment strategies. Vaccines of different countries are in clinical and preclinical trials and the repurposed-drugs are providing to find out a positive result against COVID-19. The report is an analysis of published information focusing on treatment options including vaccination, drug-therapy, cytokines, therapeutic antibodies. Most of the vaccine’s development strategies and drugs target the surface structural spike glycoprotein or S-protein, the major inducer of pathogenic responses. Here, it is reviewed the features of SARS-CoV-2 and the global current status of therapeutic, and vaccine development for the prevention and effective treatment of COVID-19.

Lycorine, a non-nucleoside RNA dependent RNA polymerase inhibitor, as potential treatment for emerging coronavirus infections

Background: Highly effective novel treatments need to be developed to suppress emerging coronavirus (CoV) infections such as COVID-19. The RNA dependent RNA polymerase (RdRp) among the viral proteins is known as an effective antiviral target. Lycorine is a phenanthridine Amaryllidaceae alkaloid isolated from the bulbs of Lycoris radiata (L'Hér.) Herb. and has various pharmacological bioactivities including antiviral function.Purpose: We investigated the direct-inhibiting action of lycorine on CoV's RdRp, as potential treatment for emerging CoV infections.Methods: We examined the inhibitory effect of lycorine on MERS-CoV, SARS-CoV, and SARS-CoV-2 infections, and then quantitatively measured the inhibitory effect of lycorine on MERS-CoV RdRp activity using a cell-based reporter assay. Finally, we performed the docking simulation with lycorine and SARS-CoV-2 RdRp.Results: Lycorine efficiently inhibited these CoVs with IC50 values of 2.123 ± 0.053, 1.021 ± 0.025, and 0.878 ± 0.022 μM, respectively, comparable with anti-CoV effects of remdesivir. Lycorine directly inhibited MERS-CoV RdRp activity with an IC50 of 1.406 ± 0.260 μM, compared with remdesivir's IC50 value of 6.335 ± 0.731 μM. In addition, docking simulation showed that lycorine interacts with SARS-CoV-2 RdRp at the Asp623, Asn691, and Ser759 residues through hydrogen bonding, at which the binding affinities of lycorine (−6.2 kcal/mol) were higher than those of remdesivir (−4.7 kcal/mol).Conclusions: Lycorine is a potent non-nucleoside direct-acting antiviral against emerging coronavirus infections and acts by inhibiting viral RdRp activity; therefore, lycorine may be a candidate against the current COVID-19 pandemic.

COVID-19, an emerging coronavirus infection: Advances and prospects in designing convalescent sera , immunotherapeutics and therapeutics

COVID-19, an emerging coronavirus infection: Advances and prospects in designing convalescent sera , immunotherapeutics and therapeutics

A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity.

Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19) are emerging zoonotic diseases caused by coronavirus (CoV) infections. The viral RNA-dependent RNA polymerase (RdRp) has been suggested as a valuable target for antiviral therapeutics because the sequence homology of CoV RdRp is highly conserved. We established a cell-based reporter assay for MERS-CoV RdRp activity to test viral polymerase inhibitors. The cell-based reporter system was composed of the bicistronic reporter construct and the MERS-CoV nsp12 plasmid construct. Among the tested nine viral polymerase inhibitors, ribavirin, sofosbuvir, favipiravir, lamivudine, zidovudine, valacyclovir, vidarabine, dasabuvir, and remdesivir, only remdesivir exhibited a dose-dependent inhibition. Meanwhile, the Z-factor and Z′-factor of this assay for screening inhibitors of MERS-CoV RdRp activity were 0.778 and 0.782, respectively. Ribavirin and favipiravir did not inhibit the MERS-CoV RdRp activity, and non-nucleoside HCV RdRp inhibitor, dasabuvir, partially inhibited MERS-CoV RdRp activity. Taken together, the cell-based reporter assay for MERS-CoV RdRp activity confirmed remdesivir as a direct inhibitor of MERS-CoV RdRp in cells. A cell-based MERS-CoV RdRp activity reporter assay is reliable and accurate for screening MERS-CoV RdRp-specific inhibitors. It may provide a valuable platform for developing antiviral drugs for emerging CoV infections.

COVID-19, an Emerging Coronavirus Infection: Current Scenario and Recent Developments – An Overview

During December 2019, a novel coronavirus virus (2019-nCov) emerged in China, which posed an International Public Health Emergency in a couple of weeks, and very recently attained the position of a very high-risk category by World Health Organization (WHO). This virus was named the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV), and the disease referred to as Coronavirus Disease-19 (COVID-19). Till March 8, 2020, the virus has claimed the lives of nearly 3,600 humans out a total of approximately 110,000 confirmed cases affected by this infection. The present editorial is a brief overview highlighting the most salient features and facts with regards to COVID-19, an emerging coronavirus infection, its causative virus (SARS-CoV-2), the current worldwide scenario, recent developments and currently ongoing progresses to contain and control this disease which have now spread to more than 100 countries across the globe. Of note, worldwide researchers and various health agencies are all together doing their best to halt the spread of this virus and avoid any possible pandemic situation to be faced, which otherwise would threaten the lives of millions of human beings.

Public Tourist Bus, Tourist Bus Driver, and COVID-19 Infection: A Note.

Dear Editor, COVID‑19 is a new emerging coronavirus infection that becomes global public health problem in year 2020. This disease first appeared in China in 2019 before the spreading worldwide occurred. At present (3 March 2020), more than 45 countries around the world are already affected by COVID‑19 Thailand is a tropical country in Southeast where COVID‑19 was first imported from China and became the second country in the world that disease emerged.

Need for integrated surveillance at human-animal interface for rapid detection & response to emerging coronavirus infections using One Health approach.

Need for integrated surveillance at human-animal interface for rapid detection & response to emerging coronavirus infections using One Health approach.

Epigenetic Landscape during Coronavirus Infection.

Coronaviruses (CoV) comprise a large group of emerging human and animal pathogens, including the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) strains. The molecular mechanisms regulating emerging coronavirus pathogenesis are complex and include virus–host interactions associated with entry, replication, egress and innate immune control. Epigenetics research investigates the genetic and non-genetic factors that regulate phenotypic variation, usually caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. Epigenetic modifications, such as histone modifications, DNA methylation, chromatin remodeling, and non-coding RNAs, function as important regulators that remodel host chromatin, altering host expression patterns and networks in a highly flexible manner. For most of the past two and a half decades, research has focused on the molecular mechanisms by which RNA viruses antagonize the signaling and sensing components that regulate induction of the host innate immune and antiviral defense programs upon infection. More recently, a growing body of evidence supports the hypothesis that viruses, even lytic RNA viruses that replicate in the cytoplasm, have developed intricate, highly evolved, and well-coordinated processes that are designed to regulate the host epigenome, and control host innate immune antiviral defense processes, thereby promoting robust virus replication and pathogenesis. In this article, we discuss the strategies that are used to evaluate the mechanisms by which viruses regulate the host epigenome, especially focusing on highly pathogenic respiratory RNA virus infections as a model. By combining measures of epigenome reorganization with RNA and proteomic datasets, we articulate a spatial-temporal data integration approach to identify regulatory genomic clusters and regions that play a crucial role in the host’s innate immune response, thereby defining a new viral antagonism mechanism following emerging coronavirus infection.

Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd

In 2012 in Jordan, infection by a novel coronavirus (CoV) caused the first known cases of Middle East respiratory syndrome (MERS). MERS-CoV sequences have since been found in a bat and the virus appears to be enzootic among dromedary camels across the Arabian Peninsula and in parts of Africa. The majority of human cases have occurred in the Kingdom of Saudi Arabia (KSA). In humans, the etiologic agent, MERS-CoV, has been detected in severe, mild and influenza-like illness and in those without any obvious signs or symptoms of disease. MERS is often a lower respiratory tract disease associated with fever, cough, breathing difficulties, pneumonia that can progress to acute respiratory distress syndrome, multiorgan failure and death among more than a third of those infected. Severe disease is usually found in older males and comorbidities are frequently present in cases of MERS. Compared to SARS, MERS progresses more rapidly to respiratory failure and acute kidney injury, is more often observed as severe disease in patients with underlying illnesses and is more often fatal. MERS-CoV has a broader tropism than SARS-CoV, rapidly triggers cellular damage, employs a different receptor and induces a delayed proinflammatory response in cells. Most human cases have been linked to lapses in infection prevention and control in healthcare settings, with a fifth of virus detections reported among healthcare workers. This review sets out what is currently known about MERS and the MERS-CoV, summarises the new phenomenon of crowd-sourced epidemiology and lists some of the many questions that remain unanswered, nearly three years after the first reported case.