Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus.

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.

Nucleocytoplasmic shuttling of the West Nile virus RNA-dependent RNA polymerase NS5 is critical to infection.

West Nile virus (WNV) is a single-stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNVKUN ) RNA-dependent RNA polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNVKUN -infected cells and NS5-transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNVKUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNVKUN , resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNVKUN replication. This study thus shows for the first time that NLS-dependent trafficking into the nucleus during infection of WNVKUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNVKUN virus production, proving the principle that inhibition of WNVKUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.

Flight traits of dengue-infected Aedes aegypti mosquitoes.

Understanding the flight behaviour of dengue-infected mosquitoes can play a vital role in various contexts, including modelling disease risks and developing effective interventions against dengue. Studies on the locomotor activity of dengue-infected mosquitoes have often faced challenges in terms of methodology. Some studies used small tubes, which impacted the natural movement of the mosquitoes, while others that used cages did not capture the three-dimensional flights, despite mosquitoes naturally flying in three dimensions. In this study, we utilised Mask RCNN (Region-based Convolutional Neural Network) along with cubic spline interpolation to comprehensively track the three-dimensional flight behaviour of dengue-infected Aedes aegypti mosquitoes. This analysis considered a number of parameters as characteristics of mosquito flight, including flight duration, number of flights, Euclidean distance, flight speed, and the volume (space) covered during flights. The accuracy achieved for mosquito detection and tracking was 98.34% for flying mosquitoes and 100% for resting mosquitoes. Notably, the interpolated data accounted for only 0.31%, underscoring the reliability of the results. Flight traits results revealed that exposure to the dengue virus significantly increases the flight duration (p-value 0.0135 × 10-3) and volume (space) covered during flights (p-value 0.029) whilst decreasing the total number of flights compared to uninfected mosquitoes. The study did not observe any evident impact on the Euclidean distance (p-value 0.064) and speed (p-value 0.064) of Aedes aegypti. These results highlight the intricate relationship between dengue infection and the flight behaviour of Aedes aegypti, providing valuable insights into the virus transmission dynamics. This study focused on dengue-infected Aedes aegypti mosquitoes; future research can explore the impact of other arboviruses on mosquito flight behaviour.

Culex-Transmitted Diseases: Mechanisms, Impact, and Future Control Strategies using Wolbachia.

Mosquitoes of the Culex genus are responsible for a large burden of zoonotic virus transmission globally. Collectively, they play a significant role in the transmission of medically significant diseases such as Japanese encephalitis virus and West Nile virus. Climate change, global trade, habitat transformation and increased urbanisation are leading to the establishment of Culex mosquitoes in new geographical regions. These novel mosquito incursions are intensifying concerns about the emergence of Culex-transmitted diseases and outbreaks in previously unaffected areas. New mosquito control methods are currently being developed and deployed globally. Understanding the complex interaction between pathogens and mosquitoes is essential for developing new control strategies for Culex species mosquitoes. This article reviews the role of Culex mosquitos as vectors of zoonotic disease, discussing the transmission of viruses across different species, and the potential use of Wolbachia technologies to control disease spread. By leveraging the insights gained from recent successful field trials of Wolbachia against Aedes-borne diseases, we comprehensively discuss the feasibility of using this technique to control Culex mosquitoes and the potential for the development of next generational Wolbachia-based control methods.

EggCountAI: a convolutional neural network-based software for counting of Aedes aegypti mosquito eggs.

BACKGROUND: Mosquito-borne diseases exert a huge impact on both animal and human populations, posing substantial health risks. The behavioural and fitness traits of mosquitoes, such as locomotion and fecundity, are crucial factors that influence the spread of diseases. In existing egg-counting tools, each image requires separate processing with adjustments to various parameters such as intensity threshold and egg area size. Furthermore, accuracy decreases significantly when dealing with clustered or overlapping eggs. To overcome these issues, we have developed EggCountAI, a Mask Region-based Convolutional Neural Network (RCNN)-based free automatic egg-counting tool for Aedes aegypti mosquitoes. METHODS: The study design involves developing EggCountAI for counting mosquito eggs and comparing its performance with two commonly employed tools-ICount and MECVision-using 10 microscopic and 10 macroscopic images of eggs laid by females on a paper strip. The results were validated through manual egg counting on the strips using ImageJ software. Two different models were trained on macroscopic and microscopic images to enhance egg detection accuracy, achieving mean average precision, mean average recall, and F1-scores of 0.92, 0.90, and 0.91 for the microscopic model, and 0.91, 0.90, and 0.90 for the macroscopic model, respectively. EggCountAI automatically counts eggs in a folder containing egg strip images, offering adaptable filtration for handling impurities of varying sizes. RESULTS: The results obtained from EggCountAI highlight its remarkable performance, achieving overall accuracy of 98.88% for micro images and 96.06% for macro images. EggCountAI significantly outperformed ICount and MECVision, with ICount achieving 81.71% accuracy for micro images and 82.22% for macro images, while MECVision achieved 68.01% accuracy for micro images and 51.71% for macro images. EggCountAI also excelled in other statistical parameters, with mean absolute error of 1.90 eggs for micro, 74.30 eggs for macro, and a strong correlation and R-squared value (0.99) for both micro and macro. The superior performance of EggCountAI was most evident when handling overlapping or clustered eggs. CONCLUSION: Accurate detection and counting of mosquito eggs enables the identification of preferred egg-laying sites and facilitates optimal placement of oviposition traps, enhancing targeted vector control efforts and disease transmission prevention. In future research, the tool holds the potential to extend its application to monitor mosquito feeding preferences.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Nuclear localisation of West Nile virus NS5 protein modulates host gene expression.

The involvement of the nucleus during flavivirus infection has been observed in only a small number of cases and can be limited to primarily two viral proteins; the structural protein C and the RNA polymerase NS5. Previously we observed that by blocking nuclear transport, WNV strain Kunjin (WNVKUN) replication is severely affected and through mutation of the identified NLS in WNVKUN NS5 protein. In this study, we interrogated the potential nuclear functions of WNVKUN NS5 has on the host transcriptome, by means of RNA sequencing (RNAseq). In a direct comparison between wild type and mutant NS5, it can also be determined that the nuclear translocation of NS5 results in a significant down-regulation of host genes involved in the innate immune response. When compared to published RNAseq data from WNV infection, many of these genes were overlapping indicting the role of NS5 induced transcription during infection.

Neutrophils mediate HIV-specific antibody-dependent phagocytosis and ADCC.

There is growing evidence to support the role of Fc-mediated effector functions, such as Antibody-Dependent Cellular cytotoxicity (ADCC) and Antibody-Dependent Phagocytosis (ADP) in the protection and control of HIV. The RV144 trial and other recent HIV vaccine studies have highlighted the importance of ADCC responses in protection against HIV. The role of neutrophils, the most abundant leukocyte in the blood, has not been thoroughly evaluated for Fc-mediated effector functions to HIV. We optimized HIV-specific neutrophil ADCC and Antibody-Dependent Neutrophil Phagocytosis (ADNP) assays using freshly isolated primary human neutrophils from blood. We also developed methods to study ADP using the neutrophil-like HL-60 cell line. We found that neutrophils mediate both HIV-specific ADP and ADCC responses. In vitro, neutrophil-mediated ADCC responses peaked at 4 h, much faster than primary NK cell or monocyte-mediated responses. We detected a wide range of responses in the ADNP, HL-60 mediated ADP and ADCC across a cohort of 41 viremic antiretroviral therapy naïve HIV positive subjects. HL-60 and Neutrophil-mediated ADP and ADCC responses correlated well with each other, suggesting that they measure overlapping functions. The ADNP and HL-60 ADP inversely correlated with HIV viral load, suggesting that these antibody-mediated neutrophil-based assays should prove useful in dissecting HIV-specific immunity.

The IMPORTance of the Nucleus during Flavivirus Replication.

Flaviviruses are a large group of arboviruses of significant medical concern worldwide. With outbreaks a common occurrence, the need for efficient viral control is required more than ever. It is well understood that flaviviruses modulate the composition and structure of membranes in the cytoplasm that are crucial for efficient replication and evading immune detection. As the flavivirus genome consists of positive sense RNA, replication can occur wholly within the cytoplasm. What is becoming more evident is that some viral proteins also have the ability to translocate to the nucleus, with potential roles in replication and immune system perturbation. In this review, we discuss the current understanding of flavivirus nuclear localisation, and the function it has during flavivirus infection. We also describe-while closely related-the functional differences between similar viral proteins in their nuclear translocation.