Biomolecules continually sample alternative conformations. Consequently, even the most energetically favored ground conformational state has a finite lifetime. Here, we show that, in addition to the 3D structure, the lifetime of a ground conformational state determines its biological activity. Using hydrogen-deuterium exchange nuclear magnetic resonance spectroscopy, we found that Zika virus exoribonuclease-resistant RNA (xrRNA) encodes a ground conformational state with a lifetime that is ~10 5 –10 7 longer than that of canonical base pairs. Mutations that shorten the apparent lifetime of the ground state without affecting its 3D structure decreased exoribonuclease resistance in vitro and impaired virus replication in cells. Additionally, we observed this exceptionally long-lived ground state in xrRNAs from diverse infectious mosquito-borne flaviviruses. These results demonstrate the biological significance of the lifetime of a preorganized ground state and further suggest that elucidating the lifetimes of dominant 3D structures of biomolecules may be crucial for understanding their behaviors and functions.

Despite numerous studies highlighting the higher transmissibility of the African Zika virus (ZIKV) lineage compared to the Asian lineage in mosquito vectors, little is known about how the viruses interact with different tissues during the early steps of mosquito infection. To address this gap, we aimed to characterize intra-host infection barriers by combining a tissue-level monitoring of infection using plaque assays and a novel quantitative analysis of single-cell level infection kinetics by in situ immunofluorescent staining. Our results revealed that, in Aedes aegypti , an African ZIKV strain exhibited a higher replication rate across various tissues than an Asian ZIKV strain. This difference was potentially due to a higher virus production in individual cells, faster spread within tissues, or a combination of both factors. Furthermore, we observed that higher bloodmeal titers resulted in a faster viral spread to neighboring cells suggesting that intra-host infection dynamics depend on inoculum size. We also identified a significant bottleneck during midgut infection establishment for both ZIKV lineages, with only a small percentage of the virus population successfully initiating infection. Finally, the in situ immunofluorescent staining technique enabled the examination of virus infection characteristics in different cell types and revealed heterogeneity in viral replication. Together, these findings demonstrate that differences in intra-host infection kinetics underlie differential transmissibility between African and Asian ZIKV lineages. This information could serve as a starting point to further investigate the underlying mechanisms and ultimately inform the development of alternative control strategies. Importance The recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency.

ABSTRACT The Zika virus (ZIKV) outbreak in Brazil between 2015 and 2016 was associated with an increased prevalence of severe congenital malformations, including microcephaly. Notably, the distribution of microcephaly cases was not uniform across the country, with a disproportionately higher incidence recorded in the Northeast region (NE). Our previous work demonstrated that saxitoxin (STX), a toxin ubiquitously present in the drinking water reservoirs of the NE, exacerbated the damaging effects of ZIKV on the developing brain. In the present study, we hypothesized that STX’s impact might vary among different neural cell types. Our experimental observations suggest that exposure to STX potentiates the neurotoxic effect of Zika Virus (ZIKV) on human neuronal cells. However, while ZIKV infection demonstrated severe impacts on astrocytes and neural stem cells (NSCs), the addition of STX did not exacerbate these effects. We observed that neurons subjected to STX exposure were more prone to apoptosis and displayed a higher number of ZIKV-infected cells. These findings suggest that STX exacerbates the harmful effects of ZIKV on neurons, thereby providing a plausible explanation for the heightened severity of ZIKV-induced congenital malformations observed in Brazil’s NE. This study underscores the importance of understanding the interactive effects of environmental toxins and infectious pathogens on neural development, with potential implications for public health policies and interventions.

Zika virus (ZIKV) and Dengue Virus (DENV) share a lot of similarities, being both flaviviruses and neglected tropical diseases that cause a worldwide burden that is increasing with climate change. Studying the interactions of cell recptors and viruses is essential to further the knowledge and development of ways to prevent and control flaviviral diseases. An important interaction occurs between the envelopeof the two viruses and a protein on the surface of immune cells, called DC-SIGN. We describe this interaction in a structural comparison involving homology modeling of these proteins, guided docking based on deposited crystallography, molecular dynamics of the docked complexes and for the decomposition of interacting residues we employed an analysis of the surface area accessible by the Generalized Bourne method. The region of interest for the discussion is predominantly electropositive in the DENV envelope, but when comparing the same region in the ZIKV envelope, we can observe that there is a decrease in charges, not to the point of making the region electronegative as a whole, but enough for the surroundings of the interaction region to become neutral with electronegative trends. These results suggest better interaction of ZIKV with the DC-SIGN receptor, particularly in the CRD portion

Improving antigen presentation is crucial for the success of immunization strategies. Yeasts are classically used as biofactories to produce recombinant proteins and are efficient vehicles for the delivery of vaccine antigens, besides present adjuvants properties. Despite the absence of epidemic outbreaks, several vaccine approaches continue to be developed for Zika virus infection. These prophylactic strategies are fundamental given the severity of clinical manifestations, mainly due to viral neurotropism. The present study aimed to evaluate in vivo the immune response induced by P. pastoris recombinant strains displaying epitopes of the Envelope (ENV) and NS1 ZIKV proteins. Intramuscular immunization with heat-attenuated yeast enhanced the secretion of IL-6, TNF-α, and IFN-γ, besides activation of CD4+ and CD8+ T cells, in BALB/c mice. P. pastoris displaying ENV epitopes induced a more robust immune response, increasing immunoglobulin production, especially IgG isotypes. Both proposed vaccines showed the potential to induce immune responses without adverse effects, confirming the safety of administering P. pastoris as a vaccine vehicle. Here we demonstrated, for the first time, the evaluation of a vaccine against ZIKV based on a multiepitope construct, using yeast as a vehicle, reinforcing the applicability of P. pastoris as a whole yeast cell vaccine.

The Zika virus (ZIKV) epidemic brought new discoveries regarding arboviruses, especially flaviviruses, as ZIKV was described as sexually and vertically transmitted. The latter shows severe consequences for the embryo/fetus, such as congenital microcephaly and deficiency of the neural system, currently known as Congenital ZIKV Syndrome (CZS). To better understand ZIKV dynamics in trophoblastic cells present in the first trimester of pregnancy (BeWo, HTR-8, and control cell HuH-7), an experiment of viral kinetics was performed for African MR766 low passage and Asian-Brazilian IEC ZIKV lineages. The results were described independently, and demonstrated that the three placental cells lines are permissive and susceptible to ZIKV. We noticed cytopathic effects that are typical in vitro viral infection in BeWo and HTR-8. Regarding kinetics, MR766lp showed peaks of viral loads in 24 and 48 hpi for all cell types tested, as well as marked cells death after peak production. On the other hand, HTR-8 lineage inoculated with ZIKV-IEC exhibited increased viral production in 144 hpi, with a peak between 24 and 96 hpi. Furthermore, IEC had peak variations of viral production for BeWo in 144 hpi. Both cells types continued alive during the process of viral replication. Considering such in vitro results, the hypothesis that maternal-fetal transmission is probably a way of virus transmission between the mother and the embryo/fetus is maintained.

Background Chikungunya and Zika are both arboviruses transmitted through the Aedes mosquitoes, which are ectothermic, leading to seasonal outbreak patterns of virus infections in the human population. Mathematical models linked with mosquito trap data, human case data, or both, have proven to be powerful tools for understanding the transmission dynamics of arboviral diseases. However, while predictive models should consider a variety of features in the environment, vectors, and hosts, it is not clear which aspects are essential to assist with short-term forecasting. Methodology We consider four simple models with various assumptions, including mosquito dynamics, temperature impacts, or both, and apply each model to forecast the Chikungunya and Zika outbreaks of nine different regions in French Polynesia. We use standard statistical criteria to compare the accuracy of each model in predicting the magnitude of the outbreak to select the most appropriate model to use as an alert system for arbovirus infections. Moreover, by calibrating our “best model”, we estimate biologically meaningful parameter values to explore the commonality and difference between Chikungunya and Zika epidemics. Conclusions We show that incorporating the mosquito population dynamics in the arbovirus transmission model is essential for accurate arbovirus case prediction. In addition, such enhancement in the accuracy of prediction is more obvious for the Chikungunya data than the Zika data, suggesting that mosquito dynamics play a more important role in Chikungunya transmission than Zika transmission. In contrast, incorporating the effects of temperature may not be necessary for past outbreaks in French Polynesia. With the well-calibrated model, we observe that the Chikungunya virus has similar but slightly higher transmissibility than the Zika virus in most regions. The best-fit parameters for the mosquito model suggest that Chikungunya has a relatively longer mosquito infectious period and a higher mosquito-to-human transmission rate. Further, our findings suggest that universal vector control plans will help prevent future Zika outbreaks. In contrast, targeted control plans focusing on specific mosquito species could benefit the prevention of Chikungunya outbreaks.

Perineuronal nets (PNNs) form a specialized extracellular matrix that regulates neuronal activity. Their formation early in the postnatal period is regulated by neuronal signaling and glial activation raising concerns that the long-term neurological sequelae ascribed to perinatal viral infections could be mediated by altered PNN formation. Previously, we developed neonatal zika virus (ZIKV) infection model where mice have lifelong neurological sequelae despite resolving the acute infection. Here, we demonstrate that neonatal ZIKV infection results in a reduction of PNN formation during the acute disease with significant reduction in Wisteria floribunda agglutinin (WFA) staining and increased aggrecan and brevican degradants. Following resolution of infection, the level of WFA staining as well as levels of aggrecan and brevican in brains normalize, but there is increased frequency of abnormal or immature PNN. In adults, the impact of the perinatal infection subsides and PNN levels and morphology are not different from control mice. Of note, the reduction in PNN formation during acute infection was associated with increased expression of MMP12 and MMP19, but not MMP9, ADAMTS-4 or ADAMTS-5. Together our findings indicate that infection at the time of PNN development interferes with PNN formation, but nets can reform once the infection and inflammation subside.

Background: Primary human cytomegalovirus (HCMV) infection can be especially serious in pregnant women and cause severe consequences for newborns. We aimed to determine which metabolites, among the thousands in human sera, are strongly correlated with serious outcomes for pregnant women with HCMV. During the early stage of the pandemic, it was predicted that COVID-19 severity is correlated with serum D-xylose/xylitol levels, which has now been confirmed in two studies. Methods: Between July 2022 and February 2023, four antiviral assays were performed by Virology Research Services Ltd. to test the antiviral activities of D-xylose, a small bioactive molecule, alone or in combination with insulin against HCMV in HFF, HIV-1 NL4-3 in HeLa TZM-bl, and ZIKV African strain and SARS-CoV-2 England strain separately in VeroE6, with incubation durations of 5 days, 48 h, 48 h and 72 h post-infection, respectively. Ganciclovir, 3′-Azido-3′-deoxythymidine (AZT), monensin and remdesivir were used as controls. An immunofluorescence method was used for readouts of all viruses except for SARS-CoV-2, for which CPE was considered. Cytotoxicity was assessed by an MTT assay. Results: The results show that D-xylose exhibits antiviral activities against SARS-CoV-2, ZIKV, HCMV and HIV-1 in vitro, with preliminary selectivity indices of 11.6, 1.6, 2.1 and 2.6, respectively. Thus, D-xylose is more effective over a broader range of concentrations against HCMV and SARS-CoV-2 than against ZIKV and HIV-1. The EC50 value (in mM) for different assays must be placed in perspective; the average serum concentration of D-xylose in a healthy person is approximately 1.8 Mm. Conclusions: The properties of the cell-layer HS stimulated by D-xylose and especially unfractionated heparin (UFH)—another antiviral compound against these viruses—combined with these results challenge the conclusion that HS promotes viral infections. Trial Registration Not Applicable.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can influence adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we show that similarities in bacterial community structures when given identical microbiomes between different genetic backgrounds of Ae. aegypti was dependent on the source microbiome, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti . We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Human pathogenic flaviviruses pose a significant health concern and vaccination is the most effective instrument to control their circulation. How pre-existing immunity to antigenically related viruses modulates immunization outcome remains poorly understood. In this study, we evaluated the effect of vaccination against tick-borne encephalitis virus (TBEV) on the epitope immunodominance and immunogenicity of the yellow fever 17D vaccine (YF17D) in a cohort of 250 human vaccinees. Following YF17D vaccination, all study participants seroconverted and generated protective neutralizing antibody titers. At day 28, TBEV pre-immunity did not affect the polyclonal neutralizing response which largely depended on the IgM fraction. We found that sera from TBEV-immunized individuals enhanced YF17D vaccine virus infection via antibody-dependent enhancement (ADE). Upon vaccination, individuals with TBEV pre-immunity had higher concentrations of cross-reactive IgG antibodies with limited neutralizing capacity against YF17D whereas vaccinees without prior flavivirus exposure showed a non-cross-reacting response. Using a set of recombinant YF17D envelope protein mutants displaying different epitopes, we identified quaternary epitopes as the primary target of neutralizing antibodies. Sequential immunizations redirected the IgG response towards the pan-flavivirus fusion loop epitope (FLE) with the potential to mediate enhancement of dengue and Zika virus infections whereas TBEV naïve individuals elicited an IgG response directed towards neutralizing epitopes without an enhancing effect. We propose that the YF17D vaccine effectively conceals the FLE and primes a neutralizing IgG response in individuals with no prior flavivirus exposure. In contrast, the response in TBEV-experienced recipients favors weakly-neutralizing, cross-reactive epitopes potentially increasing the risk of severe dengue and Zika disease due to ADE.

We present compelling evidence for the existence of an evolutionary adaptive response to viral agents such as SARS-CoV-2, that results in the human in vivo biosynthesis of a family of compounds with potential antiviral activity. Using nuclear magnetic resonance (NMR) spectroscopy, we detected a characteristic spin-system motif indicative of the presence of an extended panel of urinary and serum metabolites during the acute viral phase. The structure of eight of nucleoside analogues was elucidated (six of which have not previously been reported in human urine), and subsequently confirmed by total-synthesis and matrix spiking. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ and IL-10, suggest an association with the viperin enzyme contributing to an endogenous innate immune defense mechanism against viral infection.

Understanding the population dynamics of an infectious disease requires linking within-host dynamics and between-host transmission in a quantitative manner, but this is seldom done in practice. Here a simple phenomenological model for viral dynamics within a host is linked to between-host transmission by assuming that the probability of transmission is related to log viral titre. Data from transmission experiments for two viral diseases of livestock, foot-and- mouth disease virus in cattle and swine influenza virus in pigs, are used to parameterise the model and, importantly, test the underlying assumptions. The model allows the relationship between within-host parameters and transmission to be determined explicitly through their influence on the individual reproduction number and generation time. Furthermore, these critical within-host parameters (time and level of peak titre, viral growth and clearance rates) can be computed from more complex within-host models, raising the possibility of assessing the impact of within-host processes on between-host transmission in a more detailed quantitative manner. Author summary For a pathogen to be able to transmit between hosts it must replicate to a sufficiently high level within an infected host. Because of this linking the dynamics of a pathogen within a host to transmission between hosts is important for understanding an infectious disease and its control. In this study I develop a simple mathematical model for the within-host dynamics and combine it with a model relating the probability of transmission to the level of the pathogen. I use the model derive explicit relationships between parameters related to the within-host dynamics, such as viral growth and clearance rates, and summary transmission measures, such as the reproduction number and generation time. I test the assumptions in the underlying model and estimate parameters using data from transmission experiments for two important viral diseases, foot-and-mouth disease virus in cattle and swine influenza virus in pigs. Identifying the critical within host parameters that influence transmission allows the impact of within-host processes on between-host transmission to be investigated in a more detailed quantitative manner.

Zika virus (ZIKV) is spread by mosquitos, sexual intercourse and vertically during pregnancy. The 2015–2016 ZIKV epidemic infected millions in the Americas and resulted in thousands of infants born with malformations. Though the clusters of severe birth defects have subsided since 2017, ZIKV transmission remains a concern throughout Latin America and the Caribbean. Travel-associated and sexually-transmitted Zika, therefore, remain potential routes of transmission for women of reproductive age and their partners. This is particularly true for communities with high immigrant and foreign-born populations in Central Brooklyn, New York. Limited information has been collected on the perception by this population of ZIKV and how high-risk women engage in preventive practices. Using a survey adapted from the WHO, we assessed engagement in mosquito-related preventive practices while traveling. Data from 483 respondents on knowledge and perceived ZIKV concern, along with demographics as correlates of engagement in preventive practices were collected using a convenience sample between September 2020 and January 2021. Data were collected via a multipronged approach using social media in REDCap. Our findings show that being white/not Hispanic, pregnant, knowledgeable and concerned about ZIKV, and having enough information about ZIKV were all significantly associated with an increased likelihood of engaging in preventive practices while traveling. Multivariable logistic modeling revealed that knowledge was significantly associated with an increased likelihood of engaging in preventive practices while traveling (AOR = 1.90, 1.28–2.83). These findings underscore the importance of directing tailored health education efforts to vulnerable populations.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that persistently infects patients, enters protected brain, placental, and testicular compartments, is sexually transmitted, and causes fetal microcephaly in utero . ZIKV persistently infects brain microvascular endothelial cells (hBMECs) that form the blood-brain-barrier and Sertoli cells that form testicular barriers, establishing reservoirs that enable viral dissemination. ZIKV persistence requires inhibiting interferon (IFN) responses that direct viral clearance. We found that ZIKV induces IFN-β and IFN-λ in hBMECs but post-transcriptionally inhibits IFN-β/λ expression. IFNβ/λ mRNAs contain AU-rich elements (AREs) in their 3’ untranslated regions which regulate protein expression through interactions with ARE binding proteins (ARE-BPs). We found that ZIKV infection of primary hBMECs induces the expression of the ARE-BP tristetraprolin (TTP) and that TTP is a novel regulator of endothelial IFN secretion. In hBMECs, TTP knockout (KO) increased IFN-β/λ 1 mRNA abundance and IFN-β/λ 1 secretion in response to ZIKV infection and inhibited viral persistence. In contrast, TTP expression dramatically reduced IFN-β/λ 1 secretion in hBMECs. IFN-β/λ 1 mRNA stability was not significantly altered by TTP and is consistent with TTP inhibition of IFN-β/λ 1 translation. TTP is similarly induced by ZIKV infection of Sertoli cells, and like hBMECs, TTP expression or KO inhibited or enhanced IFN-β/λ mRNA levels, respectively. These findings reveal a mechanism for ZIKV induced TTP to promote viral persistence in hBMECs and Sertoli cells by post-transcriptionally regulating IFN-β/λ secretion. Our results demonstrate a novel role for virally induced TTP in regulating IFN secretion in barrier cells that normally restrict viral persistence and spread to protected compartments. Importance Our findings define a novel role for ZIKV induced TTP expression in regulating IFN-β/λ production in primary hBMECs and Sertoli cells. These cells comprise key physiological barriers subverted by ZIKV to access brain and testicular compartments and serve as reservoirs for persistent replication and dissemination. We demonstrate for the first time that the ARE binding protein TTP is virally induced and post-transcriptionally regulates IFN-β/λ secretion. In ZIKV infected hBMEC and Sertoli cells, TTP knockout increased IFN-β/λ secretion, while TTP expression blocked IFN-β/λ secretion. The TTP directed blockade of IFN secretion permits ZIKV spread and persistence in hBMECs and Sertoli cells and may similarly augment ZIKV spread across IFN-λ protected placental barriers. Our work highlights the importance of post-transcriptional ZIKV regulation of IFN expression and secretion in cells that regulate viral access to protected compartments and defines a novel mechanism of ZIKV regulated IFN responses which facilitate neurovirulence and sexual transmission.

Globally, health promotion measures have been undertaken in preventing the emergence and spread of coronavirus disease 2019 (COVID-19). However, whether these measures influence public awareness and behaviors is unclear and evidence is limited in particular in low-and-middle income country. We conducted an online survey among internet users in Bangladesh to understand the status and attributes of their knowledge, attitudes and practices towards COVID-19 during second wave of the pandemic when COVID educational information was more accessible to the public. Survey data were analyzed using descriptive statistics, Chi-square tests, and multivariate logistic regression analysis. Of 964 respondents, 40.2%, 51.5%, and 64.3% had good knowledge, confident attitudes, and proper practices towards COVID-19, respectively. The multivariate regression analysis found that the knowledge and practice scores were associated (p<0.05) with gender, age, and occupation. Females had better knowledge and practices compared to males (p<0.05). There were major gaps in awareness, attitudes, and practices among internet users in particular males and elders that needs to be addressed to control the further spread of COVID-19 infections before at least COVID-19 vaccine become accessible at population level in Bangladesh.

ABSTRACT Background Zika virus (ZIKV) has emerged as a cause of febrile illness in children and adults globally. West Asian and Middle Eastern countries have not yet experienced the widespread emergence of ZIKV. In Pakistan, detection of ZIKV antibodies have been reported. However, the validity of this data is questionable given the current understanding of flaviviral antigenic cross-reactivity. In order to determine if ZIKV is circulating in the Sindh region of Pakistan, patients presenting to healthcare centers with an acute febrile dengue-like illness were evaluated for ZIKV infection. Methods Dengue virus (DENV) screening in patients was performed using a commercial ELISA Rapid Test NS1 antigen capture test. All DENV negative samples were tested for Zika virus, using a commercial IgM capture ELISA kit. Additionally, a plaque reduction neutralization test (PRNT) was performed to measure neutralizing antibodies. Singleplex, two-step Real-time PCR using general primers and probes was performed for the detection of Zika virus nucleic acid. Results Patients with acute dengue-like illness (DLI) presenting at healthcare centers in different sites throughout the Sindh region of Pakistan were recruited. A total of 745 patient samples were tested for ZIKV via IgM ELISA and RT-PCR. Thirty-seven samples (4.9%) tested positive for ZIKV IgM without any cross-reactivity to other flaviviruses tested simultaneously. These were considered as presumptive positive for ZIKV, selected presumptive positive samples (n=20) were confirmed using PRNT50 using Vero cells. All 20 samples showed ZIKV neutralization at PRNT50. Conclusions Our study provides evidence that ZIKV is circulating in the Sindh region of Pakistan and is a probable cause of clinical dengue-like illness (DLI) cases that are seen seasonally in Pakistan.

SUMMARY In the past decade, Zika virus (ZIKV) emerged as a global public health concern. While adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of symptoms caused by this virus including microcephaly. In this study, we generated a toolkit to ectopically express Zika viral proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We use this toolkit to identify phenotypes and host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins cause scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size, and neuronal function defects. We further use this system to identify strain-dependent phenotypes that may contribute to the increased pathogenesis associated with the more recent outbreak of ZIKV in the Americas. Our work demonstrates Drosophila’s use as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

Introduction Needle-stick injuries (NSIs) are defined as the sharp point of a needle puncturing human skin. This article examines the risk and illustrates the burden of NSIs for workers in the healthcare, veterinary and research industries, and includes a sample survey population of workers in workplaces using needles. Methods For the review component of this article, PubMed and Google Scholar were queried within the date range of 1998-2022, retrieving 1,437 results. A publicly available sample population dataset was and analyzed from British Columbia (n=30) on workplace needlestick injuries. The OSHA, WHO, and NIEHS guidelines were reviewed, and the WorkSafe BC injury database was searched using FIPPA requests. Discussion Recapping remains a common practice despite decades of guidelines recommending against recapping. NSI research is underpowered and underrepresented in non-healthcare settings. NSIs lead to heightened anxiety, depression, and PTSD in workers and exposure to pathogens, toxic chemicals and permanent tissue damage. NSI annual reporting is likely an underestimate due to chronic underreporting, and the financial impact including work-loss and healthcare costs continues to rise. Current NSI prevention devices have limited uptake and thus, more affordable, versatile and efficient NSI-prevention devices are needed. Relevance Due to COVID-19, healthcare workers are at a higher risk of receiving NSIs. Emphasis on safe needle handling practices is necessary to maintain workers physical and psychological safety, to protect workers using COVID-19 PPE on long shifts, and to deliver the high volume of vaccinations required to inoculate the global population. Conclusion NSIs are detrimental to healthcare workers wellbeing, chronically underreported, and poorly surveyed. Areas of future research include determining more effective solutions to reduce NSIs, assessing the validity of NSI reporting systems, and integrating solutions with COVID-19 prevention and vaccination protocols.

Background: Mosquito borne pathogens are a threat to human health and can be difficult to detect especially when they cause a high rate of asymptomatic infections in human. The discovery that infected mosquitoes can release viruses in both their saliva and secreta gave rise to low-cost methods for the early detection of arboviruses during entomological surveillance. Methods: Here, we implemented both saliva and excreta-based entomological surveillance during the emergence of Zika virus (ZIKV) in French Guiana in 2016 by trapping mosquitoes around households of confirmed ZIKV symptomatic cases. Results: ZIKV was detected in mosquito excreta and not in mosquito saliva in one trap collection out of 85 (1.2%) throughout the survey. One female Ae. aegypti was found with a ZIKV systemic infection in the corresponding trap. The lag time between symptom onset declared by the ZIKV infected individual living in this site and ZIKV detection detected in this mosquito was one week. Conclusions: Altogether, these results highlight the potential of trapped mosquito excreta as a sensitive and cost-effective method to non-invasively detect arbovirus circulation.

Introduction: Plants of the genus Phyllanthus ( Euphorbiaceae ), the “stone breaker” has long been used in folk medicine to treat hepatitis B, an important viral disease. In this regard some species of this genus were tested against viruses as Herpes simplex virus type 1 (HSV-1), COVID and Hepatitis C virus (HCV), showing expressive inhibition in some of them, attracting attention to the genus and to its chemical constituents. Although P. brasiliensis has no traditional use as an antiviral plant, these findings urge us to consider whether P. brasiliensis , plant widely distributed in the Brazilian Amazon and its compounds have antiviral activity against Vero cell lines infected with Mayaro, Oropouche, Chikungunya, and Zika viruses. Objectives: Thus, herein we describe a study of extracts' chemical diversity, antiviral activity, and specialized metabolites detected in different parts of the Phyllanthus brasiliensis (Aubl.) Poir., Methods: In this context, liquid chromatography-mass spectrometry is a crucial analytical method to reveal the identity of many drug candidates from extracts from medicinal plants used to treat diseases, including for instance infections by viruses. Results: As result, 44 compounds were annotated by mass spectrometry-based molecular networking approach. Along, the extracts were tested against Vero cell lines infected with Mayaro, Oropouche, Chikungunya, and Zika viruses. Overall, the results revealed that this species is high in fatty acids, flavones, flavan-3-ols, and lignans. Among them, especially lignans are known to have promising antiviral properties. In this context, in vitro assays revealed potent antiviral activity against different viruses, especially lignan-rich extracts MEB (EC 50  = 0.80 µg.mL − 1 , SI = 377.59), MEL (EC 50  = 0.84 µg.mL − 1 , SI = 297.62), and HEL (EC 50  = 1.36 µg.mL − 1 , SI = 735.29) against Zika virus (ZIKV). Conclusions: The potential of lignans was also supported by in silico antiviral prediction, and Tuberculatin (a lignan) stood out with high activity score and as a strong candidate from MEL extract. Therefore, authors believe that P. brasiliensis extracts contain metabolites that could be a new kick-off point with candidates for antiviral drug development, with lignans becoming a promising trend for further virology research.

Abstract: Background: Cytidine/uridine monophosphate kinase 2 (UMP-CMP kinase 2, CMPK2) has been reported as an antiviral interferon-stimulated gene (ISG). We previously observed that the expression of CMPK2 was significantly upregulated after Zika Virus (ZIKV) infection in A549 cells. However, the role of CMPK2 in ZIKV replication remains to be determined. Methods: CMPK2 or retinoic acid inducible gene I (RIG-I) was overexpressed by plasmid transfection or knocked down by siRNA, respectively in A549 cells before the cells were infected with ZIKV. ZIKV RNA and NS1 protein were detected by RT-qPCR and Western Blot, respectively. The activation status of Jak-STAT signaling pathway was determined by the evaluation of the phosphorylation level of STAT1 (p-STAT1, by Western Blot), interferon stimulated response element (ISRE) activity (by dual luciferase assay) and interferon stimulated gene (ISG) expression (by RT-qPCR). Results: We found that ZIKV infection induced CMPK2 expression dependent on RIG-I. Overexpression of CMPK2 inhibited while CMPK2 knockdown promoted ZIKV replication. Mechanically, we found that CMPK2 inhibited ZIKV replication probably through the activation of Jak/STAT signaling pathway as shown by the increased level of p-STAT1, enhanced activity of ISRE, and the up-regulated expression of some downstream ISGs. Conclusion: ZIKV infection induced CMPK2 expression, which activated the Jak/STAT signaling pathway to inhibit ZIKV replication. Keywords: CMPK2; ZIKV; Jak/STAT signaling pathway

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N’ and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N’. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (M pro ) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibited enzyme inhibitory activity against M pro (Ki: 1-10 micM) and three of them good anti-SARS-CoV-2 infection activity in the low micromolar range (EC 50 1-12 micM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit Cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. QM/MM computer simulations assisted in the design and in elucidating the way of action. Finally, structural analysis shows, in agreement with the computer predictions, the binding mode of FGA146 and FGA147 to the active site of the protein. Our results illustrate that peptidyl nitroalkenes are potent covalent reversible inhibitors of the M pro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection becoming promising drugs against SARS-CoV-2.

The Zika virus (ZIKV) outbreaks and its co-relation with microcephaly have become a global health concern. It is primarily transmitted by a mosquito, but can also be transmitted from an infected mother to her fetus causing impairment in brain development, leading to microcephaly. However, the underlying molecular mechanism of ZIKV-induced microcephaly is poorly understood. In this study, we explored the role of ZIKV non-structural protein NS4A and NS4B in ZIKV pathogenesis in a well-characterized primary culture of human fetal neural stem cells (fNSCs). We observed that the co-transfection of NS4A and NS4B altered the neural stem cell fate by arresting proliferation and inducing premature neurogenesis. NS4A-NS4B transfection in fNSCs increased autophagy and dysregulated notch signalling. Further, it also altered the regulation of downstream genes controlling cell proliferation. Additionally, we reported that 3 methyl-adenine (3MA), a potent autophagy inhibitor, attenuated the deleterious effects of NS4A and NS4B as evidenced by the rescue in Notch1 expression, enhanced proliferation, and reduced premature neurogenesis. Our attempts to understand the mechanism of autophagy induction indicate the involvement of mitochondrial fission and ROS. Collectively, our findings highlight the novel role of NS4A and NS4B in mediating NSC fate alteration through autophagy-mediated notch degradation. The study also helps to advance our understanding of ZIKV-induced neuropathogenesis and suggests autophagy as a potential target for anti-ZIKV therapeutic intervention.