A review on RNA interference studies in Anophelines to reveal candidate genes for malaria transmission blocking vaccine.

Malaria is a major public health concern. The development of parasite-based vaccine RTS/AS01 has some therapeutic value but its lower efficacy is one of the major limitations. Mosquito-based transmission-blocking vaccines could have a higher potential for parasite inhibition within the mosquitoes. Several genes of mosquito midgut, salivary gland, hemolymph, etc. get activate in response to the Plasmodium-infected blood and helps in parasite invasion directly or indirectly inside the mosquito. The studies of such genes provided a new insight into developing the more efficient vaccines. In the field of malaria genetics research, RNAi has become an innovative strategy used to identify mosquito candidate genes for transmission-blocking vaccines. This review targeted the gene studies that have been conducted in the period 2000-2023 in different malaria vectors against different malarial parasites using the RNAi approach to reveal mosquito novel gene candidates for vaccine development.

A systematic evaluation of sample preparation and 2-D gel electrophoresis protocol for mosquito proteomic profiling.

-Mosquito act as the carrier insect to transfer pathogens into hosts for various vector-borne diseases.To identify the pathogenesis causing determinant, comprehensive knowledge of the protein expression in different tissues and physiological conditions is very important. The most widely used technique is 2-D gel electrophoresis to study the protein expression in mosquitoes. 2-D gel electrophoresis is the multistep process to resolve intact protein with similar molecular weight. It is also useful to separate post-translational modified protein, which are not distinguished through shotgun proteomic analysis. Here, we optimized the protocol for 2-D gel electrophoresis that can effectively resolve the protein in mosquitoes and some other insects, to target immunogenic protein to fight against the vector borne disease. The optimized 2-D protocol helps to resolve complex proteomic data which is very difficult to analyze in mosquitoes.The updated protocol improved the protein solubility, resolution and visualization that help in comparative analysis of protein expression.

Efficacy of maturase K and rpL20 protein extracted from C. procera leaves on Anophelesstephensi.

The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal potential on Anopheles stephensi. The whole protein was isolated using protein extraction buffer and precipitated by ammonium sulphate and larvicidal active protein was purified by the column chromatography. The homogeneity of larvicidal protein was confirmed by the SDS-PAGE. The identification of protein was done by the HPLC and LC-MS/ESI-MS. The crude protein from leaves showed 100% mortality of 3rd instar larvae of An. stephensi at the concentration of 5.5 mg/ml after 24 h of exposure. The crude protein from stem showed 25% mortality and no mortality observed was observed in seed protein. The leaves crude protein was further purified by ion exchange chromatography and eluted fractions were tested for larvicidal potential. The purified single protein fractions L2 and L3 from C. procera leaves showed 100% mortality at concentration of 0.06 mg/ml. The homogeneity of purified protein was confirmed by SDS-PAGE and two bands of 26 kDa and 15 kDa protein were observed. The peptide sequence "R.SQMLENSFLIENVMKR.L" was identified in the trypsin digested homogenous protein fraction L2 and "R.DRGSQKR.N" peptide sequence in L3 fraction by LC-MS/ESI-MS. The CprL2 peptide showed the sequence similarity with the protein maturase K and CprL3 peptide showed the sequence similarity with ribosomal protein L20 of C. procera. The conserved functional domain was also identified in both the CprL2 and CprL3 peptide. The identified proteins showed strong larvicidal efficacy at very low concentration. The identified proteins are novel and natural larvicidal agents against An. stephensi and hence can be used to control the malaria.

Mosquito gene targeted RNAi studies for vector control.

Vector-borne diseases are serious public health concern. Mosquito is one of the major vectors responsible for the transmission of a number of diseases like malaria, Zika, chikungunya, dengue, West Nile fever, Japanese encephalitis, St. Louis encephalitis, and yellow fever. Various strategies have been used for mosquito control, but the breeding potential of mosquitoes is such tremendous that most of the strategies failed to control the mosquito population. In 2020, outbreaks of dengue, yellow fever, and Japanese encephalitis have occurred worldwide. Continuous insecticide use resulted in strong resistance and disturbed the ecosystem. RNA interference is one of the strategies opted for mosquito control. There are a number of mosquito genes whose inhibition affected mosquito survival and reproduction. Such kind of genes could be used as bioinsecticides for vector control without disturbing the natural ecosystem. Several studies have targeted mosquito genes at different developmental stages by the RNAi mechanism and result in vector control. In the present review, we included RNAi studies conducted for vector control by targeting mosquito genes at different developmental stages using different delivery methods. The review could help the researcher to find out novel genes of mosquitoes for vector control.

Determination of the relative orientation between 15N-1H dipolar coupling and 1H chemical shift anisotropy tensors under fast MAS solid-state NMR.

In this work, we have proposed a proton-detected three-dimensional (3D) 15N-1H dipolar coupling (DIP)/1H chemical shift anisotropy (CSA)/1H chemical shift (CS) correlation experiment to measure the relative orientation between the 15N-1H dipolar coupling and the 1H CSA tensors under fast magic angle spinning (MAS) solid-state NMR. In the 3D correlation experiment, the 15N-1H dipolar coupling and 1H CSA tensors are recoupled using our recently developed windowless C-symmetry-based C331-ROCSA (recoupling of chemical shift anisotropy) DIPSHIFT and C331-ROCSA pulse-based methods, respectively. The 2D 15N-1H DIP/1H CSA powder lineshapes extracted using the proposed 3D correlation method are shown to be sensitive to the sign and asymmetry of the 1H CSA tensor, a feature that allows the determination of the relative orientation between the two correlating tensors with improved accuracy. The experimental method developed in this study is demonstrated on a powdered U-15N L-Histidine.HCl·H2O sample.

Transovarial transmission of mosquito-borne viruses: a systematic review.

Background: A number of mosquito-borne viruses (MBVs), such as dengue virus (DENV), zika virus (ZIKV), chikungunya (CHIKV), West Nile virus (WNV), and yellow fever virus (YFV) exert adverse health impacts on the global population. Aedes aegypti and Aedes albopictus are the prime vectors responsible for the transmission of these viruses. The viruses have acquired a number of routes for successful transmission, including horizontal and vertical transmission. Transovarial transmission is a subset/type of vertical transmission adopted by mosquitoes for the transmission of viruses from females to their offspring through eggs/ovaries. It provides a mechanism for these MBVs to persist and maintain their lineage during adverse climatic conditions of extremely hot and cold temperatures, during the dry season, or in the absence of susceptible vertebrate host when horizontal transmission is not possible. Methods: The publications discussed in this systematic review were searched for using the PubMed, Scopus, and Web of Science databases, and websites such as those of the World Health Organization (WHO) and the European Centre for Disease Prevention and Control, using the search terms "transovarial transmission" and "mosquito-borne viruses" from 16 May 2023 to 20 September 2023. Results: A total of 2,391 articles were searched, of which 123 were chosen for full text evaluation, and 60 were then included in the study after screening and removing duplicates. Conclusion: The present systematic review focuses on understanding the above diseases, their pathogenesis, epidemiology and host-parasite interactions. The factors affecting transovarial transmission, potential implications, mosquito antiviral defense mechanism, and the control strategies for these mosquito-borne viral diseases (MBVDs) are also be included in this review.

Isolation, Purification, and Characterization of Homogenous Novel Bioactive Protein from Datura stramonium Stem Exhibited Larvicidal Activity against Anopheles stephensi.

The insecticidal resistance of mosquitoes necessitates the development of a natural, safe, and plant-based method for vector control. Unfortunately, there are no effective vaccines or particular medications available to combat malaria; therefore, mosquitoes must be targeted directly. Previous studies have shown the health benefits of Datura stramonium, but its bioactive peptides or proteins are less explored. This is the first study on D. stramonium stem protein used for mosquito larval protein. The present study aimed to identify the purified mosquito larval protein from the crude extract of D. stramonium stem. Crude protein was isolated, precipitated, dialyzed, and purified by using ion-exchange chromatography, native PAGE, and HPLC. The highest larval mortality was observed at 5.5 mg/ml of crude protein concentration. Native PAGE was used for the analysis and purification of active proteins. The single homogeneous purified larvicidal protein appeared as a single band of 30 kDa by SDS-PAGE. The novel bioactive peptide was characterized by LC-MS/ESI-MS. The homology of the peptide was searched by the Mascot search engine. The database search revealed has not shown peptide similarity with D. stramonium protein, but homology with another plant Arabidopsis thaliana protein is probable for protein phosphatases. The lethal concentration of purified protein against 3rd instar larvae of Anopheles stephensi had LC50 and LC90 values of 25 µg/ml and 40 µg/ml. It has shown new insight into larvicidal activity and can be used as a new drug against malaria and other mosquito-borne diseases.

Emergence of SARS-CoV-2 New Variants and Their Clinical Significance.

COVID-19 is a respiration-related disease caused by SARS-CoV-2 and was identified in China's Wuhan city. More than 223 countries are affected by the disease worldwide. The new variants of the COVID-19 virus are causing problems, from average to life-threatening pneumonia and acute respiratory distress syndrome (ARDS). Presently, there are 170 vaccine candidates, out of which 10 have been approved by the WHO for vaccination, such as Ad26.COV2.S, Pfizer/BioNTech, COVISHIELD, Covovax, Moderna, KoviVac, and some other vaccines to combat the deadly SARS-CoV-2 infection. From all these vaccines, Pfizer/BioNTech and Moderna are showing the highest efficacy against COVID-19. These vaccines are highly efficient against COVID-19 disease, but their potentiality against new variants remains a question. COVID-19 vaccines are highly effective at preventing severe illnesses, hospitalizations, and death. The antibodies elicited by earlier infection or vaccination are the key for possible protection against SARS-CoV-2. The problem has been exacerbated by new information from Africa on the origins of the novel contagious SARS-CoV-2 strain. These new strains occur due to unique mutations in the spike protein, which modify SARS-CoV-2 transmission and infection capabilities, limiting the efficacy of the COVID-19 vaccination. Hence, there is a need to find a potential vaccine against it.

An analytical review of vector- and pathogen-based transmission-blocking vaccine for malaria control.

Malaria is a vector borne disease, considered to be one of the most serious public health problems. The present review focused on the blocking of parasite development in mosquito vectors; one broad strategy for achieving this is Transmission Blocking Vaccines (TBV). The TBVs usually rely on immunization of vertebrate hosts with molecules derived from the vector or pathogen to reduce pathogen transmission from infected to uninfected hosts. Most of the studies on the TBVs are based on the antibodies targeted against the surface antigens of sexual stages of malaria parasite, but it is meagre to develop mosquito-based vaccine in this regard. Vector-based TBVs include surface proteins that are expressed by the mosquito midgut digestive enzymes which are induced upon blood-feeding, and receptors expressed on the epithelial line of the tissue. Many proteins are reported that can act as candidates for transmission-blocking vaccines. This review aims to summarize the vector midgut-based proteins identified till date, that can block the development and maturity of sexual stages of the parasite within mosquitoes as targets for transmission-blocking vaccine development. The TBVs intervention can block transmission of different malaria parasite species in various species of mosquitoes with future application perspective worldwide.

Zika virus vertical transmission in mosquitoes: A less understood mechanism.

Zika virus disease is a great concern in different parts of the world, and it has become a Public Health Emergency of International Concern. The global pandemic of ZIKV in 2015 prompted concern among scientific community. Zika is a flavivirus of the family Flaviviridae transmitted by mosquitoes. Natural vertical transmission is an ecological strategy that arboviruses adopt to ensure their survival inside the mosquito vector during harsh conditions or interepidemic periods when horizontal transmission is difficult. ZIKV is vertically transmitted from infected females to its offspring. This review has concluded various studies regarding the vertical transmission ability of different mosquito species for ZIKV. Previously Aedes aegypti was considered to be a major vector, however Aedes albopictus and Culex quinquifasciatus are discovered to have the similar vertical transmission potential. Different studies shown that natural vertical transmission has been detected in mosquito species which are not implicated as possible vectors. It leads to the possibility that many other mosquito species may be potential ZIKV vectors.

Virtual screening and molecular dynamics simulation study of plant protease inhibitors against SARS-CoV-2 envelope protein.

Due to the outbreak of a new strain of pandemic coronavirus, there is a huge loss of economy and health. In 2021, some vaccines are recommended as emergency licensed vaccines to protect against the virus, and efforts are continuously ongoing to evaluate the vaccine safety measures for licensed vaccines. Recently, there was an increase in the cases of a new variant of coronavirus (omicron). Envelope protein plays an important role in virus packaging and assembly. If viral assembly is blocked, there is less chance of spreading the infection to another cell.In the present study, the plant protease inhibitors (PPIs) were screened against the envelope protein of SARS CoV 2. The structures were downloaded from the protein data bank. The plant protease inhibitors cystatin-I, Eravatmin, squash, Kunitz, Bowman-Birk, Alpha-amylase inhibitors, and potato serine protease inhibitors were screened and out of them Kunitz, alpha-amylase, and squash protease inhibitors have shown maximum binding energy. The molecular dynamics simulation was performed for docked complexes showing the lowest binding energy by NMA (normal mode analysis) to visualize the motion and stability of complexes. These plant-based protease inhibitors are a good target to fight against the new emerging strain of coronavirus because plant extracted compounds are natural and there is fewer side effect than synthetic compounds.

Designing spike protein (S-Protein) based multi-epitope peptide vaccine against SARS COVID-19 by immunoinformatics.

The outbreak of COVID-19 was originated from China, responsible for Several Acute Respiratory Syndrome (SARS). Scientists are forced to develop vaccine and effective drugs to control COVID-19 infection. To develop effective vaccine for SARS - COVID 19, immunoinformatics and computational approaches could helps to design successful vaccine against this biggest danger for humanity. Here we used various in - silico approaches to designed vaccine against COVID-19. To develop vaccine, we target S- protein, expressed on the virus surface plays important role in COVID-19 infection. We identified 12 B-cell, 9 T-helper and 20 Cytotoxic T-cell epitope based on criteria of selection. The predicted epitopes were link simultaneously with GPGPG & AAY linkers. The ß-defensin was used as adjuvant, linked with selected epitope by using EAAAK linker. For vaccine construct justification we analysed its immunogenicity, allergenicity and physiochemical properties. Our study revealed that vaccine was non toxic, immunogenic and antigenic in nature and covers 98.6% of world population, important for vaccine effectively. In- silico cloning was used to analyse its expression in vector. Molecular docking was performed to study the interaction of construct with TLR (TLR3, TLR4, and TLR9) molecules. The immune simulation was conducted and conformed that our vaccine constructs can induces both acquired and humoral immunity effectively against COVID-19 at very low concentration, but along with bioinformatics study we need to conduct experiment in laboratory to validate its safety and effectiveness.

A comprehensive analysis of amino-peptidase N1 protein (APN) from Anopheles culicifacies for epitope design using Immuno-informatics models.

Analysis of the Amino-peptidase N (APN) protein from Anopheles culicifacies as a vector based Transmission Blocking Vaccines (TBV) target has been considered for malaria vaccine development. Short peptides as potential epitopes for B cells and cytotoxic T cells and/or helper T cells were identified using prediction models provided by NetCTL and IEDB servers. Antigenicity determination, allergenicity, immunogenicity, epitope conservancy analysis, atomic interaction with HLA allele specific structure models and population coverage were investigated in this study. The analysis of the target protein helped to identify conserved regions as potential epitopes of APN in various Anopheles species. The T cell epitopes like peptides were further analyzed by using molecular docking to check interactions against the allele specific HLA models. Thus, we report the predicted B cell (VDERYRL) and T cell (RRYLATTQF for HLA class I and LKATFTVSI for HLA class II) epitopes like peptides from APN protein of Anopheles culicifacies (Diptera: Culicidae) for further consideration as vaccine candidates subsequent to in vitro and in vivo analysis.

Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.