Natural Virus Research Articles

Analysis and risk assessment of the introduction of small ruminant plague pathogens, sheep pox and goat pox through the territory of the West Kazakhstan region into the Russian Federation

Sheep pox and goat pox (GFP) and peste des petits ruminants (PPR) are infectious diseases of viral nature that mainly affect small ruminants. In domestic animals these are sheep and goats and in wildlife saigas, ibex, antelope and so on. These diseases are considered to be emergent infections, since they are characterized by a transboundary route of entry and spread, as well as causing enormous economic damage in small livestock due to almost 100% mortality and high lethality. Studies have shown that there is a high risk of introduction of emergent infectious diseases pathogens from the territory of West Kazakhstan region (through Kaztalovsky district) to Alexandrovo-Gaysky district of Saratov region. The risk is assessed as high, due to the fact that the largest number of susceptible small ruminants is concentrated on the territory of Kaztalovsky district, which amounts to 287640 heads. Small ruminants’ population density in this district was 15.5 heads/km2. The highest number of cattle is also observed on the territory of Kaztalovsky district (which is 117893 heads, which is of certain importance when keeping cattle and cattle together as a potential source-virus carrier of pathogens of the considered infections. Risk scoring, taking into account the criterion of availability of international routes of communication, showed 2 points out of 5. But, taking into account the fact that the interstate border is not marked and there is no control line, it entails the free movement of both wild and farm animals, in addition, the risk of uncontrolled movement and transportation of animals and livestock products through anthropogenic factor increases. Taking into account these facts, the risk can be assessed as 5 points out of 5.

The role of immune mechanisms in the pathogenesis of hemorrhagic fever with renal syndrome

Hemorrhagic fever with renal syndrome (HFRS) is an acute natural focal disease of a viral nature. Its severity is determined by hemorrhagic phenomena, cytokine storm, kidney damage with the development of acute renal failure, and the possibility of death. HFRS is caused by viruses from the family Hantaviridae. Hantaviruses are characterized by the ability to infect endothelial and epithelial cells of the renal tubules, as well as innate immunity cells (neutrophils, monocytes, dendritic cells), which leads to both direct damage to the immune system and indirect through a violation of their functions. The review analyzes the immune mechanisms in HFRS and their role in the pathogenesis of this disease.

Exploring the enzymatic activity of depolymerase gp531 from Klebsiella pneumoniae jumbo phage RaK2

Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a β-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3–8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531′s activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae.

The Role of Social Media in Health Misinformation and Disinformation During the COVID-19 Pandemic: Bibliometric Analysis.

The use of social media platforms to seek information continues to increase. Social media platforms can be used to disseminate important information to people worldwide instantaneously. However, their viral nature also makes it easy to share misinformation, disinformation, unverified information, and fake news. The unprecedented reliance on social media platforms to seek information during the COVID-19 pandemic was accompanied by increased incidents of misinformation and disinformation. Consequently, there was an increase in the number of scientific publications related to the role of social media in disseminating health misinformation and disinformation at the height of the COVID-19 pandemic. Health misinformation and disinformation, especially in periods of global public health disasters, can lead to the erosion of trust in policy makers at best and fatal consequences at worst. This paper reports a bibliometric analysis aimed at investigating the evolution of research publications related to the role of social media as a driver of health misinformation and disinformation since the start of the COVID-19 pandemic. Additionally, this study aimed to identify the top trending keywords, niche topics, authors, and publishers for publishing papers related to the current research, as well as the global collaboration between authors on topics related to the role of social media in health misinformation and disinformation since the start of the COVID-19 pandemic. The Scopus database was accessed on June 8, 2023, using a combination of Medical Subject Heading and author-defined terms to create the following search phrases that targeted the title, abstract, and keyword fields: ("Health*" OR "Medical") AND ("Misinformation" OR "Disinformation" OR "Fake News") AND ("Social media" OR "Twitter" OR "Facebook" OR "YouTube" OR "WhatsApp" OR "Instagram" OR "TikTok") AND ("Pandemic*" OR "Corona*" OR "Covid*"). A total of 943 research papers published between 2020 and June 2023 were analyzed using Microsoft Excel (Microsoft Corporation), VOSviewer (Centre for Science and Technology Studies, Leiden University), and the Biblioshiny package in Bibliometrix (K-Synth Srl) for RStudio (Posit, PBC). The highest number of publications was from 2022 (387/943, 41%). Most publications (725/943, 76.9%) were articles. JMIR published the most research papers (54/943, 5.7%). Authors from the United States collaborated the most, with 311 coauthored research papers. The keywords "Covid-19," "social media," and "misinformation" were the top 3 trending keywords, whereas "learning systems," "learning models," and "learning algorithms" were revealed as the niche topics on the role of social media in health misinformation and disinformation during the COVID-19 outbreak. Collaborations between authors can increase their productivity and citation counts. Niche topics such as "learning systems," "learning models," and "learning algorithms" could be exploited by researchers in future studies to analyze the influence of social media on health misinformation and disinformation during periods of global public health emergencies.

Racial Identity Attitudes and Vicarious Traumatization from Undue Police Violence on Anticipatory Traumatic Reaction Among Black Americans.

Although the viral nature of videos that capture violent and racialized policing of Black Americans in the United States can increase awareness, exposure to race-based violence can result in vicarious traumatization, particularly among Black Americans. The relationship between anticipatory traumatic reactions (ATRs) and racial identity attitudes is not clearly addressed in the extant body of literature. The current study addresses this research disparity by first analyzing group mean differences among Black Americans (N = 138) who were assigned to audiovisual, written, and imaginal exposure groups. The current study also used a cluster analysis of Black Americans to examine the differences between racial identity attitudes and ATRs following media exposure to undue police violence. Results from the study indicated that no differences in ATRs existed based on types of media exposure. Significant differences across three racial identity clusters existed between ATR in association with attitudes of assimilation, miseducation, self-hatred, anti-dominance, and ethnic-racial salience. Findings from the study suggest that mental health professionals should attend to racial identity attitudes as a relevant factor in how Black American clients experience the psychological impact of media exposure to undue police violence.

Populism in Context: A Cross-Country Investigation of the Facebook Usage of Populist Appeals During the 2019 European Parliament Elections

Recent scholarship demonstrated that Facebook is a fertile space for populist political communication as its unmediated and viral nature make populist appeals highly efficient in mobilizing voters. However, less attention has been paid to the way these populist messages appear through political actors’ Facebook communication, and what post- and page-level factors they are associated with. We investigate these questions in the context of the 2019 European Parliament election based on a unique cross-national dataset covering twelve European countries. In this study, we categorized 8,074 Facebook posts published on the main Facebook pages of sixty-seven parties. Our findings show that different populist appeals are used in specific ways. For example, at the post level, anti-elitism is frequently used in relation to economy, labor and social policy, and immigration; people-centric appeals are associated with labor and social policy and used when parties call for action, while out-group messages are not related to other topics beyond immigration. “Ideational populist” communication is more frequently articulated in European level and related to the topics of economy and labor and social policy. At the party level, it seems that there are still sharp differences between populist and non-populist parties in their communication.

Antibiotic utilization and resistance in diarrheagenic Escherichia coli isolated from children under 5 Years in Nakuru County: a case-control study

Background: Children under the age of 5 years continue to suffer the ravaging effects of microbial resistance. Majority of the infections associated with this age are of bacterial and viral nature. Highest mortalities in this age group are those associated with enteric and diarrheal diseases. Diarrheagenic Escherichia coli (DEC) is among the leading causes of these diseases due to their ubiquitous nature. Methods: The study adopted a case-control design and aimed at investigating antibiotic utilization and resistance in DEC strains isolated from children under 5 years in Nakuru County. A total sample size of 384 children were considered. Stool samples from anal swabs were obtained and cultured on Eosin Methylene Blue (EMB). Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method to segregate the resistant DEC isolates against amoxicillin, ampicillin, erythromycin, cefoxitin and nalidixic acid. Results: All the DEC (n=192, 100%) strains were resistant to amoxicillin, n=168, 87.5% were resistant to ampicillin, n=156, 81.3% to erythromycin n=72, 37.5% to cefoxitin and only n=64, 33.3% to nalidixic acid. Based on demographic factors, it was that observed self-medication leads among factors contributing to the observed trend in antimicrobial resistance (AMR). Other factors such the length of antibiotic use did not show any level of significance (p<0.05). Conclusions: Thus, we conclude that a conglomerate of several factors is associated with the rising cases of AMR among the DEC strains. Notably, the use of first-line antibiotics especially the β-lactams poses a critical health concern being the most resisted class of antibiotics. Therefore, the current study unravels the need to remediate the effects of AMR among the DEC strains through proper formulation and implementation of guidelines on antibiotic usage.

Pathotyping and Virulence Analysis of Xanthomonas oryzae pv. oryzae Causing Bacterial Blight of Rice in Tamil Nadu

Background-Bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice production worldwide. Resistance breeding is considered a durable, effective, and eco-friendly approach to control BB. Several BB resistance (R) genes have been identified that confer resistance against various strains of Xoo. The dynamic nature of this pathogen needs proper knowledge of pathotype composition and its virulence among the diverse Xoo strains which is imperative for designing a resistance breeding program. Methods-In this study, we conducted an extensive survey to collect BB infected samples from diverse rice-growing regions of Tamil Nadu. Further, the samples were identified as Xoo through morphological and molecular identification methods. The pathotyping and virulence profiling of Xoo isolates was carried out on a set of rice differentials consisting of 22 near-isogenic lines (NILs) of IR24 possessing different BB resistance genes under glasshouse conditions during Kharif 2021-2022. Results-Based on the disease reaction in 22 rice BB differentials (IRBB lines), the Xoo isolates were grouped into six pathotypes (I II, III, IV, V, and VI). Further, the virulence analysis revealed that the near-isogenic lines possessing single R gene and gene pyramids were susceptible to about 20-88% and 4-20% respectively to Xoo isolates. R genes and their pyramids such as (Xa7, Xa21, Xa4+xa5+Xa21, Xa4+xa5+xa13 Xa4+xa13+Xa21, xa5+xa13+Xa21, and Xa4+Xa21+xa5+Xa21) were found to be effective against all the pathotypes in Tamil Nadu. The pathotype III was predominantly distributed (40%) over the Tamil Nadu. The isolates found in the pathotypes I and II are more virulent and in the pathotypes V, VI are less virulent. The current study identified the virulence nature of diverse Xoo isolates and the effectiveness of resistance (R) genes against the Xoo isolates. Therefore, the effective R genes identified in this study could be an effective source for resistance breeding program against BB in Tamil Nadu.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells

BackgroundAs the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines.MethodsUsing CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models.ResultsWe demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection.ConclusionsOur results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like Particle (VLP) Vaccines for Cancer Immunotherapy.

Cancer vaccines are increasingly being studied as a possible strategy to prevent and treat cancers. While several prophylactic vaccines for virus-caused cancers are approved and efficiently used worldwide, the development of therapeutic cancer vaccines needs to be further implemented. Virus-like particles (VLPs) are self-assembled protein structures that mimic native viruses or bacteriophages but lack the replicative material. VLP platforms are designed to display single or multiple antigens with a high-density pattern, which can trigger both cellular and humoral responses. The aim of this review is to provide a comprehensive overview of preventive VLP-based vaccines currently approved worldwide against HBV and HPV infections or under evaluation to prevent virus-caused cancers. Furthermore, preclinical and early clinical data on prophylactic and therapeutic VLP-based cancer vaccines were summarized with a focus on HER-2-positive breast cancer.

Burkholderia pseudomallei – The malicious invader causing orbital cellulitis !!!

This case report represents an unusual presentation of Melioidosis presenting as orbital cellulitis. Orbital cellulitis is an acute infection of the eye, commonly caused by Staphylococcal, Streptococcal species and also by fungi. Here we report a case of a 57 year old male who was a diabetic and hypertensive presented with fever, protrusion of right eyeball with sudden onset of defective vision. On examination right eye visual acuity was light perception with relative afferent pupillary defect. Proptosis, chemosis and total ophthalmoplegia were present. Routine investigation revealed leucocytosis. Conjunctival swab culture was negative whereas blood culture revealed Burkholderia pseudomallei. On further work up ultrasound abdomen showed splenic abscess. Even with effective treatment with IV antibiotics patient didn’t improve and succumbed to this disease. Ocular involvement in melioidosis is rare but can lead to devastating outcomes. This case represents the highly virulent nature of this organism which rarely presents with ocular involvement as primary presentation.

Validated Molecular Marker for Downy Mildew Disease Resistance Breeding of Sunflower: A Short Review

The oomycete pathogen Plasmopara halstedii responsible for sunflower downy mildew (DM), that is a significant and important disease that greatly affects the economy. As of now, there is no non-race-specific resistance for this disease and breeders are depended on race-specific resistance to control DM disease. On the other hand, using conventional breeding procedure introgression of the DM resistance genes is a long-term task due to the highly virulent and aggressive nature of the P. halstedii pathogen. Molecular markers that can be applied at the seedling stage, offers rapid response for selection with higher precision as well as a lower cost. There are currently 36 downy mildew resistance genes (R genes), designated as Pl (Pl1-Pl36, Plhra, and PlArg, in sunflowers, each with a unique linkage group (LGs). The availability of DM resistance genomic data of sunflower, related to Single Nucleotide Polymorphisms (SNP) based markers with mine allelic diversity maximize the opportunity of utilizing Marker assisted selection (MAS) techniques for downy mildew resistance breeding. This review highlights the available genetic marker and their utilization at MAS techniques for enhancing downy mildew disease resistant breeding program of sunflowers.

Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study

Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.

Yeast and Virus-like Particles: A Perfect or Imperfect Couple?

Virus-like particles (VLPs) comprise viral structural proteins that self-assemble to form a particle similar to the native virus capsid. Since their discovery, they have been employed mainly as vaccines to prevent viral infection because they can elicit an immune response. Besides their use as vaccines, their application in cancer prevention and drug delivery is under intensive investigation. They can be produced in different systems such as bacteria, mammalian, plant, insect, and yeast cells. The main hurdle for their use is establishing a platform for production because many variables need to be considered. First, VLPs must be effective in the action for which they are constructed, depending on the nature of the VLPs. Second, the production platform must be suitable for safe and high-scale production. Yeast has been shown to be a valuable tool in VLP production, as it is able to express heterologous proteins efficiently and its manipulation is cheap and easy. Several species have been employed for this purpose. In the present review, we analyze the features of different yeast species and how they have been used to produce VLPs.

Virulence and genetic analysis of Puccinia graminis tritici in the Indian sub-continent from 2016 to 2022 and evaluation of wheat varieties for stem rust resistance.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), has re-emerged as one of the major concerns for global wheat production since the evolution of Ug99 and other virulent pathotypes of Pgt from East Africa, Europe, Central Asia, and other regions. Host resistance is the most effective, economic, and eco-friendly approach for managing stem rust. Understanding the virulence nature, genetic diversity, origin, distribution, and evolutionary pattern of Pgt pathotypes over time and space is a prerequisite for effectively managing newly emerging Pgt isolates through host resistance. In the present study, we monitored the occurrence of stem rust of wheat in India and neighboring countries from 2016 to 2022, collected 620 single-pustule isolates of Pgt from six states of India and Nepal, analyzed them on Indian stem rust differentials, and determined their virulence phenotypes and molecular genotypes. The Ug99 type of pathotypes did not occur in India. Pathotypes 11 and 40A were most predominant during these years. Virulence phenotyping of these isolates identified 14 Pgt pathotypes, which were genotyped using 37 Puccinia spp.-specific polymorphic microsatellites, followed by additional phylogenetic analyses using DARwin. These analyses identified three major molecular groups, demonstrating fewer lineages, clonality, and long-distance migration of Pgt isolates in India. Fourteen of the 40 recently released Indian wheat varieties exhibited complete resistance to all 23 Pgt pathotypes at the seedling stage. Twelve Sr genes were postulated in 39 varieties based on their seedling response to Pgt pathotypes. The values of slow rusting parameters i.e. coefficient of infection, area under disease progress curve, and infection rates, assessed at adult plant stage at five geographically different locations during two crop seasons, indicated the slow rusting behavior of several varieties. Six Sr genes (Sr2, Sr57, Sr58, Sr24, Sr31, and Sr38) were identified in 24 wheat varieties using molecular markers closely linked to these genes. These findings will guide future breeding programs toward more effective management of wheat stem rust.

New Bacteriophages with Podoviridal Morphotypes Active against Yersinia pestis: Characterization and Application Potential.

Phages of highly pathogenic bacteria represent an area of growing interest for bacterial detection and identification and subspecies typing, as well as for phage therapy and environmental decontamination. Eight new phages-YpEc56, YpEc56D, YpEc57, YpEe58, YpEc1, YpEc2, YpEc11, and YpYeO9-expressing lytic activity towards Yersinia pestis revealed a virion morphology consistent with the Podoviridae morphotype. These phages lyse all 68 strains from 2 different sets of Y. pestis isolates, thus limiting their potential application for subtyping of Y. pestis strains but making them rather promising in terms of infection control. Two phages-YpYeO9 and YpEc11-were selected for detailed studies based on their source of isolation and lytic cross activity towards other Enterobacteriaceae. The full genome sequencing demonstrated the virulent nature of new phages. Phage YpYeO9 was identified as a member of the Teseptimavirus genus and YpEc11 was identified as a member of the Helsettvirus genus, thereby representing new species. A bacterial challenge assay in liquid microcosm with a YpYeO9/YpEc11 phage mixture showed elimination of Y. pestis EV76 during 4 h at a P/B ratio of 1000:1. These results, in combination with high lysis stability results of phages in liquid culture, the low frequency of formation of phage resistant mutants, and their viability under different physical-chemical factors indicate their potential for their practical use as an antibacterial mean.

Generation of a monoclonal antibody against duck circovirus capsid protein and its potential application for native viral antigen detection.

Duck circovirus (DuCV) infection is currently recognized as an important immunosuppressive disease in commercial duck flocks in China. Specific antibodies against DuCV viral proteins are required to improve diagnostic assays and understand the pathogenesis of DuCV infection. To generate DuCV-specific monoclonal antibodies (mAbs), a recombinant DuCV capsid protein without the first 36 N-terminal amino acids was produced in Escherichia coli. Using the recombinant protein as an immunogen, a mAb was developed that reacted specifically with the DuCV capsid protein, expressed in E. coli and baculovirus systems. Using homology modeling and recombinant truncated capsid proteins, the antibody-binding epitope was mapped within the region of 144IDKDGQIV151, which is exposed to solvent in the virion capsid model structure. To assess the applicability of the mAb to probe the native virus antigen, the murine macrophage cell line RAW267.4 was tested for DuCV replicative permissiveness. Immunofluorescence and Western blot analysis revealed that the mAb recognized the virus in infected cells and the viral antigen in tissue samples collected from clinically infected ducks. This mAb, combined with the in vitro culturing method, would have widespread applications in diagnosing and investigating DuCV pathogenesis.

The presence of RNA cargo is suspected to modify the surface hydrophobicity of the MS2 phage

The surface hydrophobicity of native or engineered non-enveloped viruses and virus-like particles (VLPs) is a key parameter regulating their fate in living and artificial aqueous systems. Its modulation is mainly depending on the structure and environment of particles. Nevertheless, unexplained variations have been reported between structurally similar viruses and with pH. This indicates that some modulating factors of their hydrophobicity remain to be identified. Herein we investigate the potential involvement of RNA cargo in the MS2 phage used as non-enveloped RNA virus model, by examining the SDS-induced electrophoretic mobility shift (SEMS) determined for native MS2 virions and corresponding RNA-free VLPs at various pH. Interestingly, the SEMS of VLPs was larger and more variable from pH 5 to 9 compared to native virions. These observations are discussed in term of RNA-dependent changes in surface hydrophobicity, suggesting that RNA cargo may be a major modulator/regulator of this viral parameter.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates.

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.