Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs.

During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore.

A specific anti-COVID-19 BNT162b2 vaccine-induced early innate immune signature positively correlates with the humoral protective response in healthy and multiple sclerosis vaccine recipients.

Objectives: The very rapidly approved mRNA-based vaccines against SARS-CoV-2 spike glycoprotein, including Pfizer-BioNTech BNT162b2, are effective in protecting from severe coronavirus disease 2019 (COVID-19) in immunocompetent population. However, establishing the duration and identifying correlates of vaccine-induced protection will be crucial to optimise future immunisation strategies. Here, we studied in healthy vaccine recipients and people with multiple sclerosis (pwMS), undergoing different therapies, the regulation of innate immune response by mRNA vaccination in order to correlate it with the magnitude of vaccine-induced protective humoral responses. Methods: Healthy subjects (n = 20) and matched pwMS (n = 22) were longitudinally sampled before and after mRNA vaccination. Peripheral blood mononuclear cell (PBMC)-associated type I and II interferon (IFN)-inducible gene expression, serum innate cytokine/chemokine profile as well as binding and neutralising anti-SARS-COV-2 antibodies (Abs) were measured. Results: We identified an early immune module composed of the IFN-inducible genes Mx1, OAS1 and IRF1, the serum cytokines IL-15, IL-6, TNF-α and IFN-γ and the chemokines IP-10, MCP-1 and MIG, induced 1 day post second and third BNT162b2 vaccine doses, strongly correlating with magnitude of humoral response to vaccination in healthy and MS vaccinees. Moreover, induction of the early immune module was dramatically affected in pwMS treated with fingolimod and ocrelizumab, both groups unable to induce a protective humoral response to COVID-19 vaccine. Conclusion: Overall, this study suggests that the vaccine-induced early regulation of innate immunity is mediated by IFN signalling, impacts on the magnitude of adaptive responses and it might be indicative of vaccine-induced humoral protection.

Detection of SARS-CoV-2 viral proteins and genomic sequences in human brainstem nuclei.

Neurological manifestations are common in COVID-19, the disease caused by SARS-CoV-2. Despite reports of SARS-CoV-2 detection in the brain and cerebrospinal fluid of COVID-19 patients, it is still unclear whether the virus can infect the central nervous system, and which neuropathological alterations can be ascribed to viral tropism, rather than immune-mediated mechanisms. Here, we assess neuropathological alterations in 24 COVID-19 patients and 18 matched controls who died due to pneumonia/respiratory failure. Aside from a wide spectrum of neuropathological alterations, SARS-CoV-2-immunoreactive neurons were detected in the dorsal medulla and in the substantia nigra of five COVID-19 subjects. Viral RNA was also detected by real-time RT-PCR. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation within affected brainstem regions, and was higher when compared to controls. While the results of this study support the neuroinvasive potential of SARS-CoV-2 and characterize the role of brainstem inflammation in COVID-19, its potential implications for neurodegeneration, especially in Parkinson's disease, require further investigations.

TRK Protein Expression in Merkel Cell Carcinoma Is Not Caused by NTRK Fusions.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous malignant tumor with neuroendocrine differentiation, with a rapidly growing incidence rate, high risk of recurrence, and aggressive behavior. The available therapeutic options for advanced disease are limited and there is a pressing need for new treatments. Tumors harboring fusions involving one of the neurotrophin receptor tyrosine kinase (NTRK) genes are now actionable with targeted inhibitors. NTRK-fused genes have been identified in neuroendocrine tumors of other sites; thus, a series of 76 MCCs were firstly analyzed with pan-TRK immunohistochemistry and the positive ones with real-time RT-PCR, RNA-based NGS, and FISH to detect the eventual underlying gene fusion. Despite 34 MCCs showing pan-TRK expression, NTRK fusions were not found in any cases. As in other tumors with neural differentiation, TRK expression seems to be physiological and not caused by gene fusions.

Rapid spread of a new West Nile virus lineage 1 associated with increased risk of neuroinvasive disease during a large outbreak in northern Italy, 2022: One Health analysis.

BACKGROUND: A new strain of WNV lineage 1 (WNV - 1) emerged in the Veneto Region, northern Italy, in 2021, eight years after the last outbreak of WNV - 1 in Italy. The virus, which co-circulates with WNV-2, has become endemic in the Region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred. METHODS: Comparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from May 16th to August 21st, 2022, to determine if the high number of WNND cases was associated with WNV-1. RESULTS: As of August 21st, 2022, 222 human cases of WNV infection were confirmed by molecular testing, including 103 with fever (WNF) and 119 with WNND. WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. During the same period, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that patients with WNND were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% CI 95% 1.54 to 8.24; p = 0.0043). As observed in humans, in wild birds WNV-1 had higher infectious rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2. CONCLUSIONS: Comparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds, and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1 infected patients might have an increased risk to develop severe disease.

Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients.

Background: SARS-CoV-2 induces a spectrum of clinical conditions ranging from asymptomatic infection to life threatening severe disease. Host microRNAs have been involved in the cytokine storm driven by SARS-CoV-2 infection and proposed as candidate biomarkers for COVID-19. Methods: To discover signatures of circulating miRNAs associated with COVID-19, disease severity and mortality, small RNA-sequencing was performed on serum samples collected from 89 COVID-19 patients (34 severe, 29 moderate, 26 mild) at hospital admission and from 45 healthy controls (HC). To search for possible sources of miRNAs, investigation of differentially expressed (DE) miRNAs in relevant human cell types in vitro. Results: COVID-19 patients showed upregulation of miRNAs associated with lung disease, vascular damage and inflammation and downregulation of miRNAs that inhibit pro-inflammatory cytokines and chemokines, angiogenesis, and stress response. Compared with mild/moderate disease, patients with severe COVID-19 had a miRNA signature indicating a profound impairment of innate and adaptive immune responses, inflammation, lung fibrosis and heart failure. A subset of the DE miRNAs predicted mortality. In particular, a combination of high serum miR-22-3p and miR-21-5p, which target antiviral response genes, and low miR-224-5p and miR-155-5p, targeting pro-inflammatory factors, discriminated severe from mild/moderate COVID-19 (AUROC 0.88, 95% CI 0.80-0.95, p<0.0001), while high leukocyte count and low levels of miR-1-3p, miR-23b-3p, miR-141-3p, miR-155-5p and miR-4433b-5p predicted mortality with high sensitivity and specificity (AUROC 0.95, 95% CI 0.89-1.00, p<0.0001). In vitro experiments showed that some of the DE miRNAs were modulated directly by SARS-CoV-2 infection in permissive lung epithelial cells. Conclusions: We discovered circulating miRNAs associated with COVID-19 severity and mortality. The identified DE miRNAs provided clues on COVID-19 pathogenesis, highlighting signatures of impaired interferon and antiviral responses, inflammation, organ damage and cardiovascular failure as associated with severe disease and death.

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain, northern Italy, June 2022.

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and - in the following days - in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.

Case Report: The Carotid Body in COVID-19: Histopathological and Virological Analyses of an Autopsy Case Series.

Various authors have hypothesized carotid body (CB) involvement in Coronavirus Disease 2019 (COVID-19), through direct invasion or indirect effects by systemic stimuli ('cytokine storm', angiotensin-converting enzyme [ACE]1/ACE2 imbalance). However, empirical evidence is limited or partial. Here, we present an integrated histopathological and virological analysis of CBs sampled at autopsy from four subjects (2 males and 2 females; age: >70 years old) who died of COVID-19. Histopathological, immunohistochemical and molecular investigation techniques were employed to characterize Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV2) viral invasion and inflammatory reaction. SARS-CoV2 RNA was detected in the CBs of three cases through Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). In these cases, positive immunostaining for Nucleocapsid and Spike protein were also demonstrated, mainly at the level of large roundish cells consistent with type I cells, confirming direct CB invasion. In these cases, T lymphocytes showed focal aggregations in the CBs, suggestive of local inflammatory reaction. Blood congestion and microthrombosis were also found in one of the positive cases. Intriguingly, microthrombosis, blood congestion and microhaemorrages were also bilaterally detected in the CBs of the negative case, supporting the possibility of COVID-19 effects on the CB even in the absence of its direct invasion. SARS-CoV-2 direct invasion of the CB is confirmed through both immunohistochemistry and RT-PCR, with likely involvement of different cell types. We also reported histopathological findings which could be ascribed to local and/or systemic actions of SARS-CoV-2 and which could potentially affect chemoreception.

Autochthonous dengue outbreak in Italy 2020: clinical, virological and entomological findings.

BACKGROUND: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy. METHODS: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood. RESULTS: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site. CONCLUSIONS: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.

Hypopharyngeal Ulcers in COVID-19: Histopathological and Virological Analyses - A Case Report.

In coronavirus disease 2019 (COVID-19), ulcerative lesions have been episodically reported in various segments of the gastrointestinal (GI) tract, including the oral cavity, oropharynx, esophagus, stomach and bowel. In this report, we describe an autopsy case of a COVID-19 patient who showed two undiagnosed ulcers at the level of the anterior and posterior walls of the hypopharynx. Molecular testing of viruses involved in pharyngeal ulcers demonstrated the presence of severe acute respiratory syndrome - coronavirus type 2 (SARS-CoV-2) RNA, together with herpes simplex virus 1 DNA. Histopathologic analysis demonstrated full-thickness lympho-monocytic infiltration (mainly composed of CD68-positive cells), with hemorrhagic foci and necrosis of both the mucosal layer and deep skeletal muscle fibers. Fibrin and platelet microthrombi were also found. Cytological signs of HSV-1 induced damage were not found. Cells expressing SARS-CoV-2 spike subunit 1 were immunohistochemically identified in the inflammatory infiltrations. Immunohistochemistry for HSV1 showed general negativity for inflammatory infiltration, although in the presence of some positive cells. Thus, histopathological, immunohistochemical and molecular findings supported a direct role by SARS-CoV-2 in producing local ulcerative damage, although a possible contributory role by HSV-1 reactivation cannot be excluded. From a clinical perspective, this autopsy report of two undiagnosed lesions put the question if ulcers along the GI tract could be more common (but frequently neglected) in COVID-19 patients.

Antibody Response to the BNT162b2 mRNA COVID-19 Vaccine in Subjects with Prior SARS-CoV-2 Infection.

Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.

SARS-CoV-2 Infection and Disease Modelling Using Stem Cell Technology and Organoids.

In this Review, we briefly describe the basic virology and pathogenesis of SARS-CoV-2, highlighting how stem cell technology and organoids can contribute to the understanding of SARS-CoV-2 cell tropisms and the mechanism of disease in the human host, supporting and clarifying findings from clinical studies in infected individuals. We summarize here the results of studies, which used these technologies to investigate SARS-CoV-2 pathogenesis in different organs. Studies with in vitro models of lung epithelia showed that alveolar epithelial type II cells, but not differentiated lung alveolar epithelial type I cells, are key targets of SARS-CoV-2, which triggers cell apoptosis and inflammation, while impairing surfactant production. Experiments with human small intestinal organoids and colonic organoids showed that the gastrointestinal tract is another relevant target for SARS-CoV-2. The virus can infect and replicate in enterocytes and cholangiocytes, inducing cell damage and inflammation. Direct viral damage was also demonstrated in in vitro models of human cardiomyocytes and choroid plexus epithelial cells. At variance, endothelial cells and neurons are poorly susceptible to viral infection, thus supporting the hypothesis that neurological symptoms and vascular damage result from the indirect effects of systemic inflammatory and immunological hyper-responses to SARS-CoV-2 infection.

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region.

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

Tuberculosis-Associated MicroRNAs: From Pathogenesis to Disease Biomarkers.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the most lethal infectious diseases with estimates of approximately 1.4 million human deaths in 2018. M. tuberculosis has a well-established ability to circumvent the host immune system to ensure its intracellular survival and persistence in the host. Mechanisms include subversion of expression of key microRNAs (miRNAs) involved in the regulation of host innate and adaptive immune response against M. tuberculosis. Several studies have reported differential expression of miRNAs during active TB and latent tuberculosis infection (LTBI), suggesting their potential use as biomarkers of disease progression and response to anti-TB therapy. This review focused on the miRNAs involved in TB pathogenesis and on the mechanism through which miRNAs induced during TB modulate cell antimicrobial responses. An attentive study of the recent literature identifies a group of miRNAs, which are differentially expressed in active TB vs. LTBI or vs. treated TB and can be proposed as candidate biomarkers.

Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells.

West Nile virus (WNV) and Usutu virus (USUV) are genetically related neurotropic mosquito-borne flaviviruses, which frequently co-circulate in nature. Despite USUV seeming to be less pathogenic for humans than WNV, the clinical manifestations induced by these two viruses often overlap and may evolve to produce severe neurological complications. The aim of this study was to investigate the effects of WNV and USUV infection on human induced pluripotent stem cell-derived neural stem cells (hNSCs), as a model of the neural progenitor cells in the developing fetal brain and in adult brain. Zika virus (ZIKV), a flavivirus with known tropism for NSCs, was used as the positive control. Infection of hNSCs and viral production, effects on cell viability, apoptosis, and innate antiviral responses were compared among viruses. WNV displayed the highest replication efficiency and cytopathic effects in hNSCs, followed by USUV and then ZIKV. In these cells, both WNV and USUV induced the overexpression of innate antiviral response genes at significantly higher levels than ZIKV. Expression of interferon type I, interleukin-1ß and caspase-3 was significantly more elevated in WNV- than USUV-infected hNSCs, in agreement with the higher neuropathogenicity of WNV and the ability to inhibit the interferon response pathway.

New avenues for therapeutic discovery against West Nile virus.

Introduction: West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus, which is endemic in many countries, especially in Europe and in North America, where the virus has increased its activity in the recent years. No vaccines nor antiviral drugs are available for the prevention and treatment of WNV infection in humans.Areas covered: This review article describes viral and host targets that have been addressed by anti-WNV drug discovery studies and summarizes the most relevant anti-WNV candidate compounds identified so far, focusing on those showing antiviral efficacy in in vivo models and broad-spectrum anti-flavivirus activity.Expert opinion: The most promising anti-WNV drug candidates target conserved enzymatic motifs in viral NS3 protease and NS5 polymerase and are effective against different flaviviruses. Targeting host factors required for viral infection and replication and modulation of host innate antiviral response are also promising approaches, which may lead to the development of compounds with broad-spectrum antiviral activity, a desirable feature for an antiviral drug.

Modelling Neurotropic Flavivirus Infection in Human Induced Pluripotent Stem Cell-Derived Systems.

Generation of human induced pluripotent stem cells (hiPSCs) and their differentiation into a variety of cells and organoids have allowed setting up versatile, non-invasive, ethically sustainable, and patient-specific models for the investigation of the mechanisms of human diseases, including viral infections and host-pathogen interactions. In this study, we investigated and compared the infectivity and replication kinetics in hiPSCs, hiPSC-derived neural stem cells (NSCs) and undifferentiated neurons, and the effect of viral infection on host innate antiviral responses of representative flaviviruses associated with diverse neurological diseases, i.e., Zika virus (ZIKV), West Nile virus (WNV), and dengue virus (DENV). In addition, we exploited hiPSCs to model ZIKV infection in the embryo and during neurogenesis. The results of this study confirmed the tropism of ZIKV for NSCs, but showed that WNV replicated in these cells with much higher efficiency than ZIKV and DENV, inducing massive cell death. Although with lower efficiency, all flaviviruses could also infect pluripotent stem cells and neurons, inducing similar patterns of antiviral innate immune response gene expression. While showing the usefulness of hiPSC-based infection models, these findings suggest that additional virus-specific mechanisms, beyond neural tropism, are responsible for the peculiarities of disease phenotype in humans.

Clinical and virological findings in patients with Usutu virus infection, northern Italy, 2018.

BackgroundUsutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years.AimTo describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018.MethodsFrom 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel.ResultsEight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2.ConclusionsClinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

In silico approaches to Zika virus drug discovery.

INTRODUCTION: After the WHO declared Zika virus (ZIKV) as a public health emergency of international concern, intense research for the development of vaccines and drugs has been undertaken, leading to the development of several candidates. Areas covered: This review discusses the developments achieved so far by computational methods in the discovery of candidate compounds targeting ZIKV proteins, i.e. the envelope and capsid structural proteins, the NS3 helicase/protease, and the NS5 methyltransferase/RNA-dependent RNA polymerase. Expert opinion: Research for effective drugs against ZIKV is still in a very early discovery phase. Notwithstanding the intense efforts for the development of new drugs and the identification of several promising candidates by using different approaches, including computational methods, so far only a few candidates have been experimentally tested. An important caveat of anti-flavivirus drug development is represented by the difficult of reproducing the in vivo microenvironment of the replication complex, which may lead to discrepancies between in vitro results and experimental evaluation in vivo. Moreover, anti-ZIKV drugs have the additional requirement of an excellent safety profile in pregnancy and ability to diffuse to different tissues, including the central nervous system, the testis, and the placenta.