The fetal lineage is susceptible to Zika virus infection within days of fertilization.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Tracking the Replication-Competent Zika Virus with Tetracysteine-Tagged Capsid Protein in Living Cells.

Real-time imaging of viruses in living cells considerably facilitates the study of virus-host interactions. However, generating a fluorescently labeled recombinant virus is challenging, especially for Zika virus (ZIKV), which causes microcephaly in neonates. The monocistronic nature of the ZIKV genome represents a major challenge for generating a replication-competent genetically engineered ZIKV suitable for real-time imaging. Here, we generated a fluorescent ZIKV by introducing the biarsenical tetracysteine (TC) tag system. After separately inserting the TC tag at six sites in the capsid protein, we found that only when we inserted the TC tag at the site of amino acids 27/28 (AA27/28, or TC27) could the genetically engineered ZIKV be rescued. Importantly, the TC27 ZIKV is characterized as replication and infection competent. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the dynamic nuclear import behavior of the capsid protein. In addition, using the single-particle tracking technology, we acquired real-time imaging evidence that ZIKV moved along the cellular filopodia and entered into the cytoplasm via endocytosis. Thus, we provide a feasible strategy to generate a replication-competent TC-tagged ZIKV for real-time imaging, which should greatly facilitate the study of ZIKV-host interactions in living cells. IMPORTANCE Zika virus (ZIKV) is the mosquito-borne enveloped flavivirus that causes microcephaly in neonates. While real-time imaging plays a critical role in dissecting viral biology, no fluorescent, genetically engineered ZIKV for single-particle tracking is currently available. Here, we generated a replication-competent genetically engineered ZIKV by introducing the tetracysteine (TC) tag into its capsid protein. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the nuclear import behavior of the capsid protein and the endocytosis process of single ZIKV particle. Taken together, these results demonstrate a fluorescent labeling strategy to track the ZIKV-host interactions at both the protein level and the viral particle level. Our replication-competent TC27 ZIKV should open an avenue to study the ZIKV-host interactions and may provide applications for antiviral screening.

A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Nuclear localization of Zika virus NS5 contributes to suppression of type I interferon production and response.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which caused an unprecedented epidemic in Latin America. Among all viral non-structural proteins in flavivirus, NS5 is the most highly conserved and has multiple crucial functions, including participating in viral RNA replication and suppressing host innate immunity. Although ZIKV NS5 prominently localizes in the nucleus during infection, its specific nuclear localization signal (NLS), and its role in viral replication and pathogenesis remain controversial. Here, we identified aa 11-90 and aa 370-406 regions that contain NLSs, which are critical for nuclear localization of ZIKV NS5. Further experiments demonstrated that nuclear localization of ZIKV NS5 predominantly participates in suppression of interferon regulatory factor 3 (IRF3)-mediated activation of type I IFN (IFN-I) transcription and inhibition of IFN-I downstream response independent of its effect on signal transducers and activators of transcription 2 (STAT2) degradation. These results suggest that subcellular localization of NS5 is important for its function on innate immune suppression, which provides new insight into ZIKV pathogenesis.

Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Epilepsy in children with Congenital Zika Syndrome: A systematic review and meta-analysis.

OBJECTIVES: To estimate the overall frequency of epilepsy in children with congenital Zika syndrome (CZS) and describe the profile of seizures and the response rate to anti-epileptic treatment in this group of patients. METHODS: A systematic review and meta-analysis were conducted following the Cochrane Handbook and preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. PubMed/MEDLINE, Scopus, Cochrane Library, SciELO, and LILACS were searched until June 23, 2020. Observational studies that evaluated the frequency of epilepsy in children diagnosed with CZS according to international criteria were included in the study. RESULTS: Fourteen studies evaluating 903 patients diagnosed with CZS were pooled in a meta-analysis. All studies were conducted in Brazil, with reports published between 2016 and 2020, and included children diagnosed with CSZ from 0 to 40 months of age. The overall rate of epilepsy in children diagnosed with CZS was estimated at 60% (95% confidence interval [CI] 0.51-0.68). The studies included in this review show that the frequency of epilepsy in patients with CSZ varies with age, with higher rates in older children. Epileptic spasms was the primary type of seizure observed in this group, followed by focal and generalized crisis. The response rate to anti-epileptic drugs was considerably low, ranging from 20% of seizure control in the first year and 30% in the second year. SIGNIFICANCE: Children with CZS presented a high cumulative incidence of epilepsy episodes with increased severity and a low response to anti-epileptic therapy, which is associated with the extensive damage caused by the Zika virus on the cortical structures of patients.

High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities.

Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids' ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.

Threshold Dynamics in a Model for Zika Virus Disease with Seasonality.

We present a compartmental population model for the spread of Zika virus disease including sexual and vectorial transmission as well as asymptomatic carriers. We apply a non-autonomous model with time-dependent mosquito birth, death and biting rates to integrate the impact of the periodicity of weather on the spread of Zika. We define the basic reproduction number [Formula: see text] as the spectral radius of a linear integral operator and show that the global dynamics is determined by this threshold parameter: If [Formula: see text] then the disease-free periodic solution is globally asymptotically stable, while if [Formula: see text] then the disease persists. We show numerical examples to study what kind of parameter changes might lead to a periodic recurrence of Zika.

Explaining Pathogenicity of Congenital Zika and Guillain-Barré Syndromes: Does Dysregulation of RNA Editing Play a Role?

Previous studies of Zika virus (ZIKV) pathogenesis have focused primarily on virus-driven pathology and neurotoxicity, as well as host-related changes in cell proliferation, autophagy, immunity, and uterine function. It is now hypothesized that ZIKV pathogenesis arises instead as an (unintended) consequence of host innate immunity, specifically, as the side effect of an otherwise well-functioning machine. The hypothesis presented here suggests a new way of thinking about the role of host immune mechanisms in disease pathogenesis, focusing on dysregulation of post-transcriptional RNA editing as a candidate driver of a broad range of observed neurodevelopmental defects and neurodegenerative clinical symptoms in both infants and adults linked with ZIKV infections. The authors collect and synthesize existing evidence of ZIKV-mediated changes in the expression of adenosine deaminases acting on RNA (ADARs), known links between abnormal RNA editing and pathogenesis, as well as ideas for future research directions, including potential treatment strategies.

Zika virus increases mind bomb 1 levels, causing degradation of pericentriolar material 1 (PCM1) and dispersion of PCM1-containing granules from the centrosome.

The centrosome is a cytoplasmic nonenveloped organelle functioning as one of the microtubule-organizing centers and composing a centriole center surrounded by pericentriolar material (PCM) granules. PCM consists of many centrosomal proteins, including PCM1 and centrosomal protein 131 (CEP131), and helps maintain centrosome stability. Zika virus (ZIKV) is a flavivirus of the family Flaviviridae whose RNA and viral particles are replicated in the cytoplasm. However, how ZIKV interacts with host cell components during its productive infection stage is incompletely understood. Here, using several primate cell lines, we report that ZIKV infection disrupts and disperses the PCM granules. We demonstrate that PCM1- and CEP131-containing granules are dispersed in ZIKV-infected cells, whereas the centrioles remain intact. We found that ZIKV does not significantly alter cellular skeletal proteins, and, hence, these proteins may not be involved in the interaction between ZIKV and centrosomal proteins. Moreover, ZIKV infection decreased PCM1 and CEP131 protein, but not mRNA, levels. We further found that the protease inhibitor MG132 prevents the decrease in PCM1 and CEP131 levels and centriolar satellite dispersion. Therefore, we hypothesized that ZIKV infection induces proteasomal PCM1 and CEP131 degradation and thereby disrupts the PCM granules. Supporting this hypothesis, we show that ZIKV infection increases levels of mind bomb 1 (MIB1), previously demonstrated to be an E3 ubiquitin ligase for PCM1 and CEP131 and that ZIKV fails to degrade or disperse PCM in MIB1-ko cells. Our results imply that ZIKV infection activates MIB1-mediated ubiquitination that degrades PCM1 and CEP131, leading to PCM granule dispersion.

Zika virus infection in chemosensory cells.

Zika virus (ZIKV) is an emerging virus belonging to the genus Flavivirus. ZIKV infection is a significant health concern, with increasing numbers of reports of microcephaly cases in fetuses and Guillain-Barré syndrome (GBS) in adults. Interestingly, chemosensory disturbances are also reported as one of the manifestations of GBS. ZIKV infects several human tissues and cell types in vitro and in vivo. However, there is no study demonstrating ZIKV infection and replication in chemosensory cells, including olfactory and taste cells. Taste papilla and olfactory cells are chemosensory receptor cells with unique histological, molecular, and physiological characteristics. Here we examined ZIKV infection (PRVABC59) in cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells in vitro, as well as in vivo mouse taste and olfactory epithelial and olfactory bulb tissues. Interestingly, while HBO cells showed resistance to ZIKV replication, hOECs were highly susceptible for ZIKV infection and replication. Further, we demonstrated the presence of ZIKV particles and expression of viral proteins in olfactory epithelium, as well as in olfactory bulb, but not in taste papillae, of immunocompromised mice (ifnar/-) infected with the PRVABC59 strain of ZIKV. These observations suggest that chemosensory cells in the olfactory neuroepithelium and olfactory bulb may be important tissues for ZIKV replication and dissemination.

Intrinsically disordered protein domains in flavivirus infection.

Intrinsically disordered protein regions are at the core of biological processes and involved in key protein-ligand interactions. The Flavivirus proteins, of viruses of great biomedical importance such as Zika and dengue viruses, exemplify this. Several proteins of these viruses have disordered regions that are of the utmost importance for biological activity. Disordered proteins can adopt several conformations, each able to interact with and/or bind to different ligands. In fact, such interactions can help stabilize a particular fold. Moreover, by being promiscuous in the number of target molecules they can bind to, these protein regions increase the number of functions that their small proteome (10 proteins) can achieve. A folding energy waterfall better describes the protein folding landscape of these proteins. A disordered protein can be thought as rolling down the folding energy cascade, in order "to fall, fold and function". This is the case of many viral protein regions, as seen in the flaviviruses proteome. Given their small size, flaviviruses are a good model system for understanding the role of intrinsically disordered protein regions in viral function. Finally, studying these viruses disordered protein regions will certainly contribute to the development of therapeutic approaches against such promising (yet challenging) targets.

Expression, Purification, and Characterization of Anti-Zika virus Envelope Protein: Polyclonal and Chicken-Derived Single Chain Variable Fragment Antibodies.

Zika virus (ZIKV) is a new and emerging virus that has caused outbreaks worldwide. The virus has been linked to congenital neurological malformations in neonates and Guillain-Barré syndrome in adults. Currently there are no effective vaccines available. As a result, there is a great need for ZIKV treatment. In this study, we developed single chain variable fragment (scFv) antibodies that target the ZIKV envelope protein using phage display technology. We first induced an immune response in white leghorn laying hens against the ZIKV envelope (E) protein. Chickens were immunized and polyclonal immunoglobulin yolk (IgY) antibodies were extracted from egg yolks. A high-level titer of anti-ZIKV_E IgY antibodies was detected using enzyme-linked immunosorbent assay (ELISA) after the third immunization. The titer persisted for at least 9 weeks. We constructed two antibody libraries that contained 5.3 × 106 and 4.5 × 106 transformants. After biopanning, an ELISA phage assay confirmed the enrichment of specific clones. We randomly selected 26 clones that expressed ZIKV scFv antibodies and classified them into two groups, short-linker and long-linker. Of these, four showed specific binding activities toward ZIKV_E proteins. These data suggest that the polyclonal and monoclonal scFv antibodies have the diagnostic or therapeutic potential for ZIKV.

Prevalence of Zika virus in blood donations: a systematic review and meta-analysis.

BACKGROUND: Transfusion-Transmitted Zika virus (TT-ZIKV) has become an emerging threat to world blood banks due to the fast spread of ZIKV epidemics and high rate of asymptomatic infections. For the risk assessment of ZIKV infection in blood products, relevant studies in blood donations or blood donors tested for ZIKV were collected and analyzed systematically. The overall prevalence of ZIKV infection were estimated through meta-analysis and potential risk factors were detected. The results will provide important clues for the protocol design of blood screening tests. METHODS: Relevant articles about the rate of ZIKV detected in blood samples were identified from PubMed, Scopus and Web Of Science using key terms search strategy until October 7, 2017. Eligible articles were screened following inclusion and exclusion criteria. Meta-analysis and subgroup analyses were performed by software R3.4.1. Overall postdonation and posttransfusion follow-ups were analyzed. RESULTS: Ten literatures (528,947 blood samples) were included for meta-analysis. The overall pooled prevalence of ZIKV (RNA and antibody) in blood donations was 1.02% (95%CI 0.36-1.99). The pooled prevalence of ZIKV RNA in blood donations was 0.85% (95%CI 0.21-1.88) less than the pooled prevalence of anti-ZIKV antibodies 1.61% (95%CI 0.03-5.21), however the difference was not statistically significant (p = 0.52). The prevalence varied significantly in different geographical regions (p < 0.001). Blood donations were more than two times likely to be infected by ZIKV in Zika epidemic period (1.37, 95%CI 0.91-1.91) than in non-epidemic period (0.61, 95%CI 0-2.55). The prevalence of anti-ZIKV antibodies (1.61, 95%CI 0.03-5.21) was almost twice as much as ZIKV nucleic acid detected in blood donations (0.85, 95%CI 0.21-1.88). However, statistically significant differences were not observed. A total of 122 ZIKV positive blood donors were followed, of which 48 (39%) reported symptoms postdonation, but none of the 13 followed recipients reported any clinical symptoms related to Zika infection posttransfusion. CONCLUSION: The pooled prevalence of Zika infection in blood donations was 1.02%. The prevalence varied greatly and reached to high-risk level in most of the situations. The results suggest that nucleic acid tests (NAT) for blood screening and pathogen reduction/inactivation technology (PRT) should be implemented in Zika-endemic areas and appropriate strategies should be designed according to different conditions. More studies are needed in the future to provide more evidence.

Zika Virus Envelope Protein and Antibody Complexes.

Zika virus (ZIKV) is a re-emerged human pathogen, belonging to a super serogroup with dengue virus. Infection of ZIKV can lead to severe congenital symptoms, such as microcephaly, in newborns and Guillain-Barré syndrome in adults. To date, no prophylactics and therapeutics are available. Flavivirus envelope (E) protein represents the major target for neutralizing antibodies, while antibody response is the key correlate of protection against ZIKV infection. A panel of monoclonal antibodies (MAbs) were found to neutralize ZIKV infection and some of them exhibited strong potential as antivirals. In this chapter, we provide a brief introduction into the history and epidemics of ZIKV. Subsequently, we describe the ZIKV envelope protein and summarize the recent progresses in MAbs development against this virus. The concomitant molecular basis for these protective MAbs is also dissected. This chapter helps to comprehensively understand the interplay between ZIKV E protein and protective MAbs.

Protein backbone flexibility pattern is evolutionarily conserved in the Flaviviridae family: A case of NS3 protease in Flavivirus and Hepacivirus.

Viruses belonging to the Flaviviridae family have been an important health concern for humans, animals and birds alike. No specific treatment is available yet for many of the viral infections caused by the members of this family. Lack of specific drugs against these viruses is mainly due to lack of protein structure information. It has been known that protein backbone fluctuation pattern is highly conserved in protein pairs with similar folds, in spite of the lack of sequence similarity. We hypothesized that this concept should also hold true for proteins (especially enzymes) of viruses included in different genera of the Flaviviridae family, as we know that the sequence similarity between them is low. Using available NS3 protease crystal structures of the Flaviviridae family, our preliminary results have shown that the Cα (i.e. backbone) fluctuation patterns are highly similar between Flaviviruses and a Hepacivirus (i.e. hepatitis C virus, HCV). This has to be validated further experimentally.

Emerging arboviruses in Rio Grande do Sul, Brazil: Chikungunya and Zika outbreaks, 2014-2016.

QUESTIONS INVESTIGATED: The recent emergence of arboviruses such as Chikungunya virus (CHIKV) and Zika virus (ZIKV) in Brazil has posed a threat to human health and to the country's economy. Outbreaks occur mainly in tropical areas; however, increasing number of cases have been observed in Rio Grande do Sul (RS), the Southernmost state; therefore, surveillance of these arboviruses is essential for public health measures. DESIGN: In this study, we analyzed 1276 samples from patients with clinically suspected arboviral diseases between 2014 and 2016. Demographic and clinical data were collected and described; cases of microcephaly associated with congenital infection were analyzed. ESSENTIAL FINDINGS: Results show that CHIKV and ZIKV entered RS in 2014 and 2015, respectively, with imported cases confirmed. Autochthonous infections occurred in 2016 for both viruses, with a total of 5 autochthonous cases for CHIKV and 44 for ZIKV. Most patients were older than 21 years; the main symptoms were fever, arthralgia, myalgia, and headache; rash, conjunctivitis, and pruritus were also reported in ZIKV cases. Three cases of congenital Zika syndrome were confirmed in our study, while another 20 cases of microcephaly associated with congenital infection were confirmed (10 positive for syphilis, 6 for toxoplasmosis and 4 for cytomegalovirus). MAIN CONCLUSIONS: Considering co-circulation of different arbovirus in RS, including Dengue virus, CHIKV, and ZIKV, and the presence of Aedes aegypti and Aedes albopictus in the area, surveillance of patients infected by these viruses contributes to the control and prevention of such diseases. Practical difficulties in diagnosing these infections are discussed.

Viral polymerase inhibitors T-705 and T-1105 are potential inhibitors of Zika virus replication.

Since 2015, 69 countries and territories have reported evidence of vector-borne Zika virus (ZIKV) transmission. Currently, there are no effective licensed vaccines or drugs available for the treatment or prevention of ZIKV infection. We tested a series of compounds for their ability to inhibit ZIKV replication in cell culture. The compounds in T-705 (favipiravir) and T-1105 were found to have antiviral activity, suggesting that these compounds are promising candidates for further development as specific antiviral drugs against ZIKV.

Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus.

BACKGROUND: Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies. METHODS: In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture. RESULTS: We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880-8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein. CONCLUSION: Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.