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1.
Braz. j. biol ; 83: e240118, 2023. tab, graf
Article in English | MEDLINE, LILACS, VETINDEX | ID: biblio-1278559

ABSTRACT

Abstract For many centuries human populations have been suffering and trying to fight with disease-bearing mosquitoes. Emerging and reemerging diseases such as Dengue, Zika, and Chikungunya affect billions of people around the world and recently has been appealing to control with chemical pesticides. Malathion (MT) is one of the main pesticides used against mosquitoes, the vectors of these diseases. This study aimed to assess cytotoxicity and mutagenicity of the malathion for the bioindicator Allium cepa L. using a multivariate and integrative approach. Moreover, an appendix table was compiled with all available literature of insecticides assessed by the Allium cepa system to support our discussion. Exposures during 48h to 0.5 mg mL-1 and 1.0 mg mL-1 MT were compared to the negative control (distilled water) and positive control (MMS solution at 10 mg L-1). The presence of chromosomal aberrations, micronuclei frequency, and mitotic index abnormalities was evaluated. Anaphase bridges were the alterations with higher incidence and presented a significantly elevated rate in the concentration of 0.5 mg mL-1, including when compared to the positive control. The integrative discriminant analysis summarizes that MT in assessed concentrations presented effects like the positive control, corroborating its potential of toxicity to DNA. Therefore, it is concluded that MT in its pure composition and in realistic concentrations used, has genotoxic potential in the biological assessment of A. cepa cells. The multivariate integrative analysis was fundamental to show a whole response of all data, providing a global view of the effect of MT on DNA.


Resumo Por muitos séculos, as populações humanas sofrem e tentam combater os mosquitos transmissores de doenças. Doenças emergentes e reemergentes como Dengue, Zika e Chikungunya afetam bilhões de pessoas em todo o mundo e, recentemente, vem apelando ao controle com pesticidas químicos. O Malation (MT) é um dos principais pesticidas usados ​​contra mosquitos, vetores dessas doenças. O objetivo deste estudo foi avaliar a citotoxicidade e a mutagenicidade do MT para o bioindicador Allium cepa L. usando uma abordagem multivariada e integrativa. Além disso, uma tabela suplementar foi compilada com toda a literatura disponível de inseticidas avaliada pelo sistema Allium cepa para apoiar nossa discussão. Exposições ao MT durante 48h a 0,5 mg mL-1 e 1,0 mg mL-1 foram comparadas a um controle negativo (água destilada) e um controle positivo (10 mg L-1 de MMS). Foram avaliadas a presença de aberrações cromossômicas, frequência de micronúcleos e anormalidades no índice mitótico. As pontes anafásicas foram as alterações com maior incidência e apresentaram uma taxa significativamente elevada na concentração de 0,5 mg mL-1, inclusive quando comparadas ao controle positivo. A análise discriminante integrativa resume que o MT nas concentrações avaliadas apresentou efeitos semelhantes ao controle positivo, corroborando seu potencial de toxicidade para o DNA. Portanto, conclui-se que o MT, em sua composição pura e nas concentrações realistas utilizadas, possui potencial genotóxico na avaliação biológica de células de A. cepa. A análise integrativa multivariada foi fundamental para mostrar uma resposta completa de todos os dados, fornecendo uma visão global do efeito da MT no DNA.


Subject(s)
Humans , Animals , Zika Virus , Zika Virus Infection , Insecticides/toxicity , DNA Damage , Chromosome Aberrations , Plant Roots , Onions , Mosquito Vectors , Malathion/toxicity , Mitotic Index
2.
Financ Res Lett ; 47: 102943, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35571984

ABSTRACT

We study the effects of epidemic diseases on corporate investment. Epidemic diseases tend to be unanticipated and exogenous to firms' decisions. Using difference-in-difference estimation strategy and a firm-level exposure to an epidemic disease measure, we find that corporate investment declines significantly following the onset of an epidemic disease. We also show that the COVID-19 pandemic has the strongest negative impact on investment when compared to the other most recent epidemic diseases.

3.
PLoS Negl Trop Dis ; 16(5): e0009805, 2022 May 12.
Article in English | MEDLINE | ID: mdl-35551520

ABSTRACT

BACKGROUND: The Hemagglutination assay (HA) is widely used in plague diagnosis, however, it has a subjective interpretation and demands high amounts of antigen and other immunobiological supplies. On the other hand, the conventional Anti-IgG ELISA is limited by the need of specific conjugates for multiple plague hosts, which leaves a gap for new diagnostic methods able to cover both the diagnosis of human cases and the epidemiological surveillance of multiple sentinel species. METHODS: We developed an ELISA Protein A-peroxidase method to detect anti-F1 antibodies across several species, including humans. To determine the cut-off and performance rates, HA results from 288 samples (81 rabbits, 64 humans, 66 rodents and 77 dogs) were used as reference. Next, we evaluated the agreement between Protein A-ELISA and Anti-IgG ELISA in an expanded sample set (n = 487). RESULTS: Optimal conditions were found with 250ng/well of F1 and 1:500 serum dilution. Protein A-ELISA showed high repeatability and reproducibility. We observed good correlation rates between the Protein A and IgG ELISAs optical densities and a higher positive/negative OD ratio for the Protein A-ELISA method. The overall sensitivity, specificity and area under the curve for Protein A-ELISA were 94%, 99% and 0.99, respectively. Similar results were observed for each species separately. In the analysis of the expanded sample set, there was a strong agreement between Protein A and IgG assays (kappa = 0.97). Furthermore, there was no cross-reaction with other common infectious diseases, such as dengue, Zika, Chagas disease, tuberculosis (humans) and ehrlichiosis, anaplasmosis and leishmaniasis (dogs). CONCLUSIONS: Altogether, the Protein A-ELISA showed high performance when compared both to HA and Anti-IgG ELISA, with a polyvalent single protocol that requires reduced amounts of antigen and can be employed to any plague hosts.

4.
Saudi J Biol Sci ; 29(4): 3015-3022, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35531165

ABSTRACT

Aedes aegypti is a principal vector for several viruses including dengue virus, chikungunya virus and zika virus. Economic burden of mosquito-borne diseases, relative failure of traditional control strategies and the resistance development against insecticides enforces towards genetic manipulation of Ae. aegypti. Hence, a key gene doublesex (Aedsx) which regulate sex differentiation and alternatively splices to form male and female specific transcripts (AedsxM and AedsxF ). CRISPR/Cas9 technique was employed to sex specifically disrupt the female-specific isoforms, AedsxF1 and AedsxF2 , both of which were shown to be expressed only in female mosquitoes. Targeting of dsxF at the developmental stage has resulted in various phenotypic anomalies of adult females. The rate of adult mutation phenotype was recorded between 29 and 37% along with anomalies of wing size, proboscis length and reduction in the sizes of pre-blood-meal and after blood-meal ovaries in dsxF1 and dsxF2 microinjected groups, respectively. These findings can be correlated with reduced fecundity rate of Go female, where AedsxF1 and AedsxF2 groups showed reduction rate in range of 23-31%. Furthermore, hatching inhibition rate of 28 to 36% was also observed in G1 generation when compared to the wildtype. Overall, these results demonstrated that AedsxF disruption has resulted in multiple female traits disruption including decreased fertility of the female that could directly or indirectly associated with reproduction and its disease transmitting abilities. All these findings suggesting that CRISPR works to alter the developmental pathways as predicted, and therefore this method potentially gives us the basis for the sex-ratio distortion system as genetic control approach for the management of this vector.

5.
Antiviral Res ; : 105330, 2022 May 06.
Article in English | MEDLINE | ID: mdl-35533778

ABSTRACT

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1-6,1-3)-ß-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 µg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 µg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 µg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

6.
J Med Entomol ; 2022 May 10.
Article in English | MEDLINE | ID: mdl-35535688

ABSTRACT

Aedes-borne viruses (ABVs) such as dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) contribute significantly to the global burden of infectious diseases, disproportionately affecting disadvantaged populations from tropical and subtropical urban areas. ABVs can be transmitted from female mosquitoes to their progeny by vertical transmission via transovarial and/or trans-egg vertical transmission and contribute to the maintenance of infected-mosquito populations year-round in endemic regions. This study describes the natural infection rate of DENV, CHIKV, and ZIKV in field-caught male Aedes (Sergentomyia) aegypti (Linnaeus) mosquitoes from Mérida, Yucatán, México, as a proxy for the occurrence of vertical virus transmission. We used indoor sequential sampling with Prokopack aspirators to collect all mosquitoes inside houses from ABV hotspots areas. Collections were performed in a DENV and CHIKV post-epidemic phase and during a period of active ZIKV transmission. We individually RT-qPCR tested all indoor collected Ae. aegypti males (1,278) followed by Sanger sequencing analysis for final confirmation. A total of 6.7% male mosquitoes were positive for ABV (CHIKV = 5.7%; DENV = 0.9%; ZIKV = 0.1%) and came from 21.0% (30/143) houses infested with males. Most ABV-positive male mosquitoes were positive for CHIKV (84.8%). The distribution of ABV-positive Ae. aegypti males was aggregated in a few households, with two houses having 11 ABV-positive males each. We found a positive association between ABV-positive males and females per house. These findings suggested the occurrence of vertical arbovirus transmission within the mosquito populations in an ABV-endemic area and, a mechanism contributing to viral maintenance and virus re-emergence among humans in post-epidemic periods.

7.
Virus Genes ; 2022 May 10.
Article in English | MEDLINE | ID: mdl-35536436

ABSTRACT

Dengue is a rapidly spreading arboviral disease that can be transmitted through any of the four types of dengue virus (DENV) serotypes. Previous studies have observed that individuals who have a pre-existing secondary infection due to a different dengue serotype, experience severe forms of this disease. During a DENV outbreak, a time-sensitive preliminary diagnosis of the origin of DENV might be useful in controlling the epidemic. Here, we developed a rapid and accurate one-step TB Green RT-PCR-based high-resolution melting (HRM) assay to identify and serotype DENV using serotyping primers based on the alignment with the E gene. This assay had a detection limit of 7.7 × 102 (DENV 1), 3.8 × 102 (DENV 2), 6.2 × 102 (DENV 3), and 1.2 × 103 (DENV 4) RNA copies/mL. No cross-reactivity with the Chikungunya, Zika, and Japanese encephalitis viruses was observed. The feasibility of using this assay for clinical diagnosis was evaluated in DENV-positive patient sera. The HRM assay and the RT-qPCR had complete matched results derived from DENV detection, including 51 serum positive and 20 serum negative. Additionally, eight DENV 2 strains in the same serotype were successfully differentiated by an HRM assay. Thus, this assay facilitated accurate detection and serotyping of DENV, along with the time-sensitive identification of the infectious focus of different DENVs.

8.
PLoS Negl Trop Dis ; 16(5): e0010426, 2022 May 10.
Article in English | MEDLINE | ID: mdl-35536870

ABSTRACT

During 2015-2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/ß receptor knockout (Ifnar-/-) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo, albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates and had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential.

9.
PLoS Negl Trop Dis ; 16(5): e0010366, 2022 May.
Article in English | MEDLINE | ID: mdl-35522620

ABSTRACT

Zika virus (ZIKV) is a mosquito-borne flavivirus and can cause neurodevelopmental disorders in fetus. As a neurotropic virus, ZIKV persistently infects neural tissues during pregnancy but the viral pathogenesis remains largely unknown. ZIKV has a positive-sense and single-stranded RNA genome, which encodes 7 non-structural (NS) proteins, participating in viral replication and dysregulation of host immunity. Like those in many other viruses, NS proteins are considered to be products evolutionarily beneficiary to viruses and some are virulence factors. However, we found that some NS proteins encoded by ZIKV genome appeared to function against the viral replication. In this report we showed that exogenously expressed ZIKV NS2A and NS4A inhibited ZIKV infection by inhibiting viral RNA replication in microglial cells and astrocytes. To understand how viral NS proteins suppressed viral replication, we analyzed the transcriptome of the microglial cells and astrocytes and found that expression of NS4A induced the upregulation of ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, ISG15 or BST2 through activating the ISGF3 signaling pathway. Upregulation of these ISGs seemed to be related to the inhibition of ZIKV replication, since the anti-ZIKV function of NS4A was partially attenuated when the cells were treated with Abrocitinib, an inhibitor of the ISGF3 signaling pathway, or were knocked down with STAT2. Aborting the protein expression of NS4A, but not its nucleic acid, eliminated the antiviral activity of NS4A effectively. Dynamic expression of viral NS proteins was examined in ZIKV-infected microglial cells and astrocytes, which showed comparatively NS4A occurred later than other NS proteins during the infection. We hypothesize that NS4A may possess intrinsic features to serve as a unique type of pathogen associated molecular pattern (PAMP), detectable by the cells to induce an innate immune response, or function with other mechanisms, to restrict the viral replication to a certain level as a negative feedback, which may help ZIKV maintain its persistent infection in fetal neural tissues.


Subject(s)
Zika Virus Infection , Zika Virus , Animals , RNA, Viral/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication , Zika Virus/physiology
10.
Cell Stem Cell ; 29(5): 810-825.e8, 2022 May 05.
Article in English | MEDLINE | ID: mdl-35523141

ABSTRACT

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.


Subject(s)
COVID-19 , Pluripotent Stem Cells , Zika Virus Infection , Zika Virus , Cell Differentiation , Female , Humans , Organoids , Placenta/metabolism , Placentation , Pluripotent Stem Cells/metabolism , Pregnancy , SARS-CoV-2 , Trophoblasts/metabolism , Zika Virus Infection/metabolism
12.
Article in English | MEDLINE | ID: mdl-35507796

ABSTRACT

BACKGROUNDS: SARS-CoV-2 infection typically presents with fever and respiratory symptoms. Besides this, COVID-19 related central and peripheral nervous system manifestations are emerging. OBJECTIVES: This study summarises the demographics, clinical profile, laboratory findings, management strategies, and outcomes in a large number of patients with COVID-19 related GBS and its variants. We also compared its clinical profile with Zika and dengue virus-related GBS. METHODS: Authors carried out a literature search up to Dec 31, 2020, in MEDLINE, PubMed, SCOPUS, Cochrane database, and Google Scholar for all published articles. RESULTS: The study identified 54 different types of articles consisting of 70 cases from 17 countries worldwide. Maximum cases 15 (21.4%) were identified from Italy, followed by USA 12 (17.1%), Spain 11 (15.7%), and Iran 10 (14.3%). The age group more than 60 years had most cases with 32 (45.7%) cases followed by age group 40-60 with cases 25 (35.7%) with male and female ratio 2. Maximum cases were treated with IVIG infusion 58 (82.9%), followed by Plasma exchange 13 (18.6%) cases. Out of 70 cases, 7 (10%) cases were manifested as Miller-Fisher syndrome. The most predominant electrodiagnostic variant was demyelinating neuropathy in 41 (73.21%) cases. The outcome reported in 67 cases was survival in 63 (90%) cases and the death of 4 (5.7%) cases. CONCLUSION: Covid 19 related GBS were reported worldwide with a better outcome. Both postinfectious or parainfectious patterns were reported. Early recognition with prompt management of GBS can prevent further severe morbidity and mortality.

13.
PLoS Negl Trop Dis ; 16(5): e0009600, 2022 May 13.
Article in English | MEDLINE | ID: mdl-35560029

ABSTRACT

BACKGROUND: Wolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseasesdengue, chikungunya, zika, and yellow fever. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia-based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity and cost issues. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the development of a LAMP assay combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers used a low-cost dye detecting system and 4 oligo nucleotide primers which can reduce the cost of analysis while the specificity is comparable to the previous methods. The detection capacity of our LAMP technique was 1.4 nM and the detection limit reduced to 2.2 fM when combined with the BIOSENSOR. Our study demonstrates that a BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification. CONCLUSIONS/SIGNIFICANCE: Our results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.

14.
Virology ; 572: 17-27, 2022 May 03.
Article in English | MEDLINE | ID: mdl-35550476

ABSTRACT

The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCSKO; B4G5KO) resulted in a marked decrease in ZIKV titers. GCSKO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCSKO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication.

15.
PLoS One ; 17(5): e0268106, 2022.
Article in English | MEDLINE | ID: mdl-35536838

ABSTRACT

INTRODUCTION: Ensuring accessible and quality health care for women and children is an existing challenge, which is further exacerbated during pandemics. There is a knowledge gap about the effect of pandemics on maternal, newborn, and child well-being. This systematic review was conducted to study maternal and child health (MCH) services utilization during pandemics (Zika, Ebola, and COVID-19) and the effectiveness of various interventions undertaken for ensuring utilization of MCH services. METHODOLOGY: A systematic and comprehensive search was conducted in MEDLINE/PubMed, Cochrane CENTRAL, Embase, Epistemonikos, ScienceDirect, and Google Scholar. Of 5643 citations, 60 potential studies were finally included for analysis. The included studies were appraised using JBI Critical appraisal tools. Study selection and data extraction were done independently and in duplicate. Findings are presented narratively based on the RMNCHA framework by World Health Organization (WHO). RESULTS: Maternal and child health services such as antenatal care (ANC) visits, institutional deliveries, immunization uptake, were greatly affected during a pandemic situation. Innovative approaches in form of health care services through virtual consultation, patient triaging, developing dedicated COVID maternity centers and maternity schools were implemented in different places for ensuring continuity of MCH care during pandemics. None of the studies reported the effectiveness of these interventions during pandemic-related health emergencies. CONCLUSION: The findings suggest that during pandemics, MCH care utilization often gets affected. Many innovative interventions were adopted to ensure MCH services. However, they lack evidence about their effectiveness. It is critically important to implement evidence-based appropriate interventions for better MCH care utilization.

16.
FASEB J ; 36 Suppl 12022 May.
Article in English | MEDLINE | ID: mdl-35552299

ABSTRACT

Viral infections continue to be the bane of human medicine, often with minimal options for treatment. Of particular concern are arboviruses, or viruses transmitted by an arthropod vector, such as a mosquito. Recently, Zika virus (ZIKV), a mosquito borne arbovirus, has emerged as a major threat to public health in many parts of the world. It is unknown what protein(s) ZIKV uses to bind to and infect cells within its mosquito vector. The receptor used by ZIKV to bind to and infect human cells has been identified as the tyrosine kinase receptor family Axl. A homolog of the Axl gene is not present within the mosquito genome. However, genes for the Down-Syndrome Cell Adhesion Molecule family members (DSCAMs) found within multiple mosquito species share key functional modular ectodomains with the human Axl gene. Here, we extracted total RNA from Culex and Aedes species of mosquitos, reverse transcribed the RNA into cDNA and then cloned the mosquito DSCAM cDNA with the highest homology to human Axl into a eukaryotic expression vector. The vector will then be transfected into a mammalian cell line void of human DSCAM and human Axl expression to determine if the mosquito DSCAM molecule is expressed as a surface protein. Finally, we will determine if ZIKV is capable of binding to the mammalian cell line that now expresses the mosquito DSCAM protein. This project will allow us to learn if ZIKV is capable of binding to the mosquito DSCAM protein. In turn, this information could lead to the possibility that the mosquito DSCAM protein allows infection by ZIKV as well. Ultimately, this research could assist vaccine development for Zika, or lead to treatments that could block ZIKA from infecting its mosquito vector.

17.
Sci Rep ; 12(1): 7810, 2022 May 12.
Article in English | MEDLINE | ID: mdl-35552469

ABSTRACT

Zika fever is an infectious disease caused by the Zika virus (ZIKV). The disease is claiming millions of lives worldwide, primarily in developing countries. In addition to vector control strategies, the most effective way to prevent the spread of ZIKV infection is vaccination. There is no clinically approved vaccine to combat ZIKV infection and curb its pandemic. An epitope-based peptide vaccine (EBPV) is seen as a powerful alternative to conventional vaccinations because of its low production cost and short production time. Nonetheless, EBPVs have gotten less attention, despite the fact that they have a significant untapped potential for enhancing vaccine safety, immunogenicity, and cross-reactivity. Such a vaccine technology is based on target pathogen's selected antigenic peptides called T-cell epitopes (TCE), which are synthesized chemically based on their amino acid sequences. The identification of TCEs using wet-lab experimental approach is challenging, expensive, and time-consuming. Therefore in this study, we present computational model for the prediction of ZIKV TCEs. The model proposed is an ensemble of decision trees that utilizes the physicochemical properties of amino acids. In this way a large amount of time and efforts would be saved for quick vaccine development. The peptide sequences dataset for model training was retrieved from Virus Pathogen Database and Analysis Resource (ViPR) database. The sequences dataset consist of experimentally verified T-cell epitopes (TCEs) and non-TCEs. The model demonstrated promising results when evaluated on test dataset. The evaluation metrics namely, accuracy, AUC, sensitivity, specificity, Gini and Mathew's correlation coefficient (MCC) recorded values of 0.9789, 0.984, 0.981, 0.987, 0.974 and 0.948 respectively. The consistency and reliability of the model was assessed by carrying out the five (05)-fold cross-validation technique, and the mean accuracy of 0.97864 was reported. Finally, model was compared with standard machine learning (ML) algorithms and the proposed model outperformed all of them. The proposed model will aid in predicting novel and immunodominant TCEs of ZIKV. The predicted TCEs may have a high possibility of acting as prospective vaccine targets subjected to in-vivo and in-vitro scientific assessments, thereby saving lives worldwide, preventing future epidemic-scale outbreaks, and lowering the possibility of mutation escape.

18.
FASEB J ; 36 Suppl 12022 May.
Article in English | MEDLINE | ID: mdl-35552900

ABSTRACT

The ongoing Covid-19 pandemic, caused by the highly transmissible SARS-CoV-2 virus, constitutes the worst global public health crisis of the past century. Although effective vaccines have been developed, administration has been slow, new variants continue to emerge, and there remains a lack of effective antivirals to treat severe cases. Thus, there remains a significant need to understand the mechanisms of SARS-CoV-2 infection and replication to identify new potential therapeutic targets. To this end, we investigated whether SARS-CoV-2 might depend on the host cell chaperone system, particularly the essential cytosolic chaperonin, CCT, to fold or assemble any of its proteins. CCT has previously been shown to be required for the replication of several other viruses including reoviruses and zika virus. We screened likely candidates among the SARS-CoV-2 proteins for co-immunoprecipitation with CCT and identified an interaction with the RNA-dependent RNA polymerase, Nsp12. To confirm Nsp12 was a substrate, we depleted cells of CCT, then transfected with Nsp12 and observed a 40% decrease in Nsp12 expression in CCT-depleted cells compared to the control. This decrease is consistent with what we have observed for other known CCT substrates. Additionally, CCT-depleted cells infected with live SARS-CoV-2 produced a 50% decrease in viral titer compared to controls, indicating that CCT is important for viral replication. A preliminary cryo-EM structure of the Nsp12-CCT complex shows a very large mass identifiable as Nsp12 inside of CCT that extends from the equatorial domains up and out through one of the two folding cavities. The structure stands to both reveal an important step in SARS-CoV-2 replication and to answer a long-standing question regarding how CCT can accommodate very large substrates.

19.
FASEB J ; 36 Suppl 12022 May.
Article in English | MEDLINE | ID: mdl-35553144

ABSTRACT

Viruses employ a variety of molecular strategies to carve out an existence in their host and to thwart host defenses. Crystal structures of viral proteins and of the host proteins deployed as molecular weapons contribute enormously to our understanding of viral pathogenesis and our efforts to combat viral infection. Mosquito-borne flaviviruses, including the dengue, Zika, yellow fever, Japanese encephalitis and West Nile viruses, cause serious human diseases in much of the world. A growing number of diverse functions has been discovered for the enigmatic virulence factor known as NS1: it is an essential cofactor in viral genome replication, a mediator of the host immune response, and a trigger of host vascular leakage. NS1 lacks precedent in the structure and sequence databases, so an accurate 3D structure was key to understanding the basis for its various functions. A high-throughput survey of expression conditions in baculovirus-infected insect cells was essential to identifying conditions for production of recombinant NS1 in its natural glycosylated, disulfide-linked form. We solved a crystal structure of West Nile virus NS1 from the anomalous scattering of the native sulfur atoms using an 18-crystal data set [1,2]. The structure revealed a previously unknown protein fold with a fundamentally dimeric architecture, and led to assigned functions for two of the three NS1 structural domains. Subsequent structures of NS1 from dengue virus and Zika viruses established the basis for its membrane association in cells and its lipid encapsidation when secreted [3]. Recently discovered functions of this remarkable virulence factor emerged in follow-up studies that built upon the crystal structures. Prime among these is the discovery that the NS1 protein, in a mouse model of dengue disease and in absence of virus, can induce the vascular leak that is a hallmark of severe dengue disease [4,5]. Clues to the molecular mechanism of NS1-induced endothelial dysfunction emerged from a study of the protection afforded by a monoclonal antibody and the structure of the Fab fragment bound to the NS1 epitope [6]. [1] Akey DL et al. & Smith JL (2014) Science 343, 881-885. [2] Akey DL et al. & Smith JL (2014) Acta Crystallogr D70, 2719-2729. [3] Brown WC et al. & Smith JL (2016) Nature Struct Molec Biol 23, 856-867. [4] Beatty PR et al. & Harris E (2015) Science Trans Med 7, 304ra141. [5] Modhiran N et al. & Young PR (2015) Science Trans Med 7, 304ra142. [6] Biering SB, Akey DL et al. Smith JL & Harris E (2021) Science 371, 194-200.

20.
FASEB J ; 36 Suppl 12022 May.
Article in English | MEDLINE | ID: mdl-35553912

ABSTRACT

Zika virus (ZIKV) infection during pregnancy can impair offspring hippocampal neurocircuit formation and cognitive function, but underlying mechanisms driving this neuronal miswiring remain unknown. During fetal development, axonal migration, pathfinding, and synaptogenesis are strongly influenced by extracellular chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs), which consist of six differentially sulfated isomers (CS-A, B (DS), C, D, E, O). The relative abundance of these CS/DS isomers can influence functions ranging from circuit plasticity (CS-C) and maturation (CS-A, B) to axonal growth attraction (CS-D) and repulsion (CS-E). Using LC-MS/MS to analyze the relative abundance of each CS/DS isomer in cortical and hippocampal tissue obtained from normal 145d fetal Macaca nemestrina nonhuman primates (NHP), a key difference between cortex and hippocampus included the presence of a less 'mature' glycan matrix environment in the latter region. This finding is supported by a 1.6-fold increase in the neuroplastic CS-C (p=0.002) and 1.7-fold increase in the neuronal attractant CS-D (p=0.0001) isomers, along with a 39%-decrease in axonal repulsive CS-E (p<0.0001), a 22%-decrease in synapse stabilizing CS-B/DS (p=0.06), and a 6%-decrease in the matrix maturing CS-A (p=0.02) isomers. The hippocampus also exhibited a 51%-decrease in the nonsulfated CS-O (p=0.0001) isomer compared to the adjacent cortex, with the resultant hypersulfation of hippocampal CS/DS-GAGs (p=0.0001) predicted to increase binding of positively charged extracellular growth factors. These differences in CS/DS-GAGs between brain regions suggest that whereas the cortex provides a matured matrix environment capable of supporting newly formed circuitry, hippocampal CS/DS-GAGs retain a developmental sulfation patterning that allows for additional neuronal migration, axon growth, and synapse formation. With this background, we then tested whether 3rd trimester congenital ZIKV infection (fetal d120-d124) in M. nemestrina influences offspring CS/DS-GAG sulfation patterns 21d later. We found that whereas no change was observed in the cortex, offspring exposed to congenital ZIKV exhibited a 33%-decrease in CS-D (p=0.01) and a 1.5x-increase in CS-O (p=0.03) in the hippocampus. This ZIKV-driven reduction of the axonal attractant CS-D can be predicted to limit hippocampal neurocircuit formation, potentially exacerbated by reduced retention of growth factors resulting from the increase in nonsulfated CS-O, which is consistent with previous evidence of reduced hippocampal neurocircuit formation after maternal ZIKV infection. Overall, these results imply that maternal ZIKV infection during late-stage fetal neurodevelopment 1) targets the immature hippocampal CS/DS-GAGs over the matured cortex, 2) identifies recoding of CS/DS-GAGs as a novel target for ZIKV infection, and 3) offers a novel and plausible mechanism to explain the deleterious impact of maternal ZIKV on offspring development.

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