Fusion Protein Gene Research Articles

Vaccination with Plasmids Encoding the Fusion Proteins D-S1, D-S1N and O-SN from SARS-CoV-2 Induces an Effective Humoral and Cellular Immune Response in Mice

Background: Next-generation vaccines against coronavirus disease 2019 (COVID-19) focus on inducing a long-lasting immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emerging variants. To achieve this, antigens other than spike proteins have been proposed, and different platforms have been evaluated. Nucleic acid-based vaccines are fundamental for this process. Preclinical data have shown that the SARS-CoV-2 nucleocapsid protein induces a protective cellular immune response, and when combined with the spike protein, the resulting humoral and cellular immune responses are effective against some SARS-CoV-2 variants. Methods: We designed a DNA vaccine against the spike and nucleocapsid proteins of SARS-CoV-2 to generate fusion proteins based on the Delta and Omicron B.5 strains. The most immunogenic regions of the spike and nucleocapsid proteins of the Delta and Omicron B strains were selected using bioinformatics. The nucleotide sequences were cloned into pcDNA3.1, and named pcDNA3.1/D-S1, pcDNA3.1/D-S1N, and pcDNA3.1/O-SN. The immunogenicity of the generated fusion proteins was evaluated by analyzing the humoral and cellular responses elicited after the immunization of BALB/c mice. Results: DNA immunization induced antibody production, neutralization activity, and IFN-γ production. The inclusion of the nucleocapsid regions in the plasmid greatly enhanced the immune response. Moreover, cross-reactions with the variants of interest were confirmed. Conclusions: Plasmids-encoding fusion proteins combining the most immunogenic regions of the spike and nucleocapsid proteins present a promising strategy for designing new and effective vaccines against SARS-CoV-2.

The combined immunization of cervical cancer therapeutic vaccine based on Listeria balanced lethal system has a significant therapeutic effect on tumor model mice.

Cervical cancer is the fourth most common cancer and the fourth leading cause of cancer death in women. Effective treatment of cervical cancer is urgently needed. Tumor therapeutic vaccine is a research hotspot in tumor immunotherapy, and the tumor therapeutic vaccine based on attenuated live bacteria carrier has clinical application prospect because of its clear action site and high safety. The bacterial balanced lethal system uses nutritional screening instead of antibiotic screening to avoid the risk of the spread of antibiotic resistance. So it is generally recognized as a green carrier. In our early research, we have constructed two cervical cancer therapeutic vaccine candidates (LM-HPVCW-1 and LI-HPVCW-1) based on Listeria balanced lethal system via transforming the nutrient-deficient strains with nutrition gene complementary plasmid. The complementary plasmid contained the nutrition complementary gene, LM dal gene, and its Amp gene was replaced with asd gene to delete the antibiotic resistance. Besides, it carried the shuffled HPV-16 E6E7 fusion protein gene. In vitro experiments showed that the plasmid carried by the candidate strain could be stably passaged and the target protein could be successfully expressed. In this study, we proved that the two candidate strains were safe in vivo and induced cellular immune response. At the same time, by establishing a tumor-bearing mouse model, it was proved that the two vaccine strains combined immunization could effectively inhibit mouse tumors. The mechanism of tumor suppression may be related to breaking the immunosuppression of tumor microenvironment and inducing CD8+ T cell infiltration. The therapeutic vaccine for cervical cancer constructed in this study is expected to be further applied in clinical practice.

Epidemiology of human metapneumovirus in Taiwan from 2013 to 2023

Human metapneumovirus (HMPV) is a member of the genus Metapneumovirus in the family Pneumoviridae of the order Mononegavirales that can cause upper and lower respiratory tract disease. This retrospective study describes the epidemiology of hMPV based on community viral surveillance results from sentinel sites across Taiwan from 2013 to 2023. A total of 114 hMPV strains were isolated and analyzed to assess viral evolution through sequencing of their fusion protein genes. This study revealed that hMPV cases occur almost year-round in Taiwan, with a peak occurring during spring (March to May). Of the 114 infected patients, 68.4% were children under 4 years old. The geographical distribution of hMPV positivity was highest in Penghu County, followed by Changhua County and Hsinchu County. The clinical symptoms of hMPV infection are nonspecific, with fever (56.1%), cough (44.7%), rhinorrhea (21.1%), and sore throat (14.9%) being the most common. However, a few patients also developed severe central nervous system symptoms (1.8%) or dyspnea (0.9%). Phylogenetic analysis revealed genetic diversity among the 114 isolated hMPV strains, with the A2 lineage (57.9%) being the most frequently observed, followed by the B2 lineage (33.3%), in the Taiwanese community from 2013 to 2023. In conclusion, hMPV causes a serious acute respiratory disease in Taiwan that should not be neglected. Further epidemiological surveillance and investigations of the clinical characteristics of hMPV should be performed continually for prevention and control of this virus.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

PCR-based detection and mutation dynamics of fusion protein gene of orthoaviula viruses sequestered during 2023 field outbreaks in Pakistan

: To isolate and detect a Newcastle disease virus in commercial poultry, Molecular characterization and phylogenetic analysis of the confirmed isolate and Multiple sequence alignment and achievement of accession numbers against our submissions in NCBI bankit.: Genetic and antigenic diversity in the fusion protein gene of New Castle disease virus strains has been recognized and the progressive changes over sequential years indicate that it is a continuously evolving virus. The current vaccines containing conventional vaccinal strains can protect birds to a certain level but do not prevent infection and virus shedding. : The partial fusion protein gene of the 14 NDV isolates during the 2023 outbreaks from different areas of Pakistan was determined and analyzed. The antigenic protein translational segment of the fusion gene nucleotide fragment was targeted with a specifically designed primer executed 202 bp size of predictable amplicon during PCR amplification. The nucleotide sequence analysis of studied isolates showed closed similarity to the NCBI bankit numbers. Phylogenetic analysis revealed that 3 isolates belong to genotype II while, 2 isolates positions near genotype VIII of class II. The 6 isolates were located near genotype XVII and only 1 was presented on genotype V branch in calss II. Mutation analysis results revealed various mutations at nucleotide intervals and even found altered amino acids during translation. The results revealed that nucleotide mutation at various positions attributes amino acid substitution that enables wild prevailing strains to evade artificial active immunity. In such a scenario Chimeric and genotype match vaccines prepared from indigenous isolates may be useful in developing candidate vaccines to prevent virus shedding and infection. Further studies are suggested at molecular level to determine the consensus amino acid sequence for virulent, mesogenic, and avirulent prevailing NDV strains.

89Zr-immunoPET-guided selection of a CD33xIL15 fusion protein optimized for antitumor immune cell activation and in vivo tumour retention in acute myeloid leukaemia.

Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.

Recent updates in pediatric diffuse glioma classification: insights and conclusions from the WHO 5th edition.

The World Health Organization (WHO) Central Nervous System (CNS) Tumors Classification 5th edition (2021) integrates both molecular and histopathological criteria for diagnosing glial tumors. This updated classification highlights significant differences between pediatric and adult gliomas in terms of molecular characteristics and prognostic implications. The 5th edition comprises a new category of pediatric-type diffuse low-grade glioma (PDLGG) and pediatric-type diffuse high-grade glioma (PDHGG), classified mainly based on genetic alterations and histopathological features. We reviewed the microscopy, diagnostic molecular pathology, and prognosis of various tumors under the categories PDLGG and PDHGG. The review also addresses the need for clarification concerning overlapping diagnostic features. PDLGG are characterized by diffuse growth, low-grade morphology, and MYB/MYBL1(MYB Proto-Oncogene Like 1) gene fusion or mitogen-activated protein kinase (MAPK) pathway alterations. In contrast, PDHGG is described by diffuse growth, high-grade morphology, and increased mitosis and often shows alterations of histone gene resulting in epigenetic alterations, which contrasts with common isocitrate dehydrogenase (IDH) mutation and epidermal growth factor receptor (EGFR) amplification seen in adult-type high-grade glioma.

Potential role of peste des petits ruminants virus in small ruminant abortions

The aim of the present study was to investigate the frequency, genetic variability, and phylogeny of the peste des petits ruminants virus (PPRV) in ovine and caprine fetuses. During 2014 and 2017, a total of 1054 embryos/fetuses were collected in Turkey. A real-time RT-PCR assay was used for the detection of the PPRV RNA. Genetic characterization and phylogenetic analysis of the PPRV field isolates were conducted by sequencing fusion (F) protein and nucleoprotein (N) gene segments. Samples were also collected from ewes (n = 83) and nanny goats (n = 3) that had aborted and whose embryos/fetuses were found to be PPRV positive. PPRV positive embryos/fetuses were also tested for the presence of Listeria monocytogenes, Campylobacter spp., Coxiella burnetii, Chlamydophila abortus, Brucella spp., akabane virus, aino virus, bluetongue virus, border disease virus, bovine viral diarrhea virus, Cache Valley virus, and Schmallenberg virus. PPRV RNA was detected in 123 (11.7 %) of the 1054 embryos/fetuses, 78 of the 83 (94 %) ewes and 3 (100 %) nanny goats. Border disease virus RNA and Chlamydophila abortus DNA were detected in 7 and 12 PPRV positive sheep fetuses, respectively, while other bacterial and viral agents were not detected. Phylogenetically, the field isolates in this study belong to lineage IV, and compared to other strains of lineage IV considered in this study, they showed 1 and 5 new amino acid substitutions in the F and N gene sequences, respectively. The results of the study suggest that PPRV plays an important role in abortion. Therefore, PPRV needs to be taken into consideration in sheep and goats abortions.

Complete genome sequence and phylogenetic analysis of a newly discovered fusagra-like virus infecting Carica papaya in Ecuador.

A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae.

Respiratory Syncytial Virus in Adult Patients at a Tertiary Care Hospital in Germany: Clinical Features and Molecular Epidemiology of the Fusion Protein in the Severe Respiratory Season of 2022/2023.

Following an interseasonal rise in mainly pediatric respiratory syncytial virus (RSV) cases in Germany in 2021, an exceptionally high number of adult cases was observed in the subsequent respiratory season of 2022/2023. The aim of this study was to compare the clinical presentation of RSV infections in the pre- and post-SARS-CoV-2 pandemic periods. Additionally, the local epidemiology of the RSV fusion protein was analyzed at a molecular genetic and amino acid level. RSV detections in adults peaked in calendar week 1 of 2023, 8 weeks earlier than the earliest peak observed in the three pre-pandemic seasons. Although the median age of the adult patients was not different (66.5 vs. 65 years), subtle differences between both periods regarding comorbidities and the clinical presentation of RSV cases were noted. High rates of comorbidities prevailed; however, significantly lower numbers of patients with a history of lung transplantation (p = 0.009), chronic kidney disease (p = 0.013), and immunosuppression (p = 0.038) were observed in the 2022/2023 season. In contrast, significantly more lower respiratory tract infections (p < 0.001), in particular in the form of pneumonia (p = 0.015) and exacerbations of obstructive lung diseases (p = 0.008), were detected. An ICU admission was noted for 23.7% of all patients throughout the study period. Sequence analysis of the fusion protein gene revealed a close phylogenetic relatedness, regardless of the season of origin. However, especially for RSV-B, an accumulation of amino acid point substitutions was noted, including in antigenic site Ø. The SARS-CoV-2 pandemic had a tremendous impact on the seasonality of RSV, and the introduction of new vaccination and immunization strategies against RSV warrants further epidemiologic studies of this important pathogen.

M-F noncoding region sequences of H1 genotype measles virus provide higher resolution for virus transmission tracing

As countries and regions move toward measles elimination, extended sequence window including noncoding region located between the matrix and fusion protein genes (M − F NCR) was considered to be used in molecular surveillance. The molecular resolution of M − F NCR was evaluated with 192 genotype H1 strains circulating during 2011–2018 in China. Phylogenetic analyses of the N450 and M − F NCR targets indicated that both two targets could confirm epi-linked outbreak, while M − F NCR target could further improve resolution of the molecular characterization: (1) it could differentiate the strains with identical N450 circulated in one county within one month of disease onset; (2) different transmission chains could be distinguished for strains with identical N450; (3) better spatial-temporal consistency with topology could be provided among sporadic cases with inconsistent N450. Accordingly, M − F NCR could be used to complement the information from N450 to address the specific questions in tracking the virus transmission chains.

Overexpression of a pearl millet WRKY transcription factor gene, PgWRKY74, in Arabidopsis retards shoot growth under dehydration and salinity-stressed conditions

Pearl millet (Cenchrus americanus) is a cereal crop that can tolerate high temperatures, drought, and low-fertility conditions where other crops lose productivity. However, genes regulating this ability are largely unknown. Transcription factors (TFs) regulate transcription of their target genes, regulate downstream biological processes, and thus are candidates for regulators of such tolerance of pearl millet. PgWRKY74 encodes a group IIc WRKY TF in pearl millet and is downregulated by drought. PgWRKY74 may have a role in drought tolerance. The objective of this study was to gain insights into the physiological and biochemical functions of PgWRKY74. Yeast one-hybrid and gel shift assays were performed to examine transcriptional activation potential and deoxyribonucleic acid (DNA)-binding ability, respectively. Transgenic Arabidopsis thaliana plants overexpressing PgWRKY74-green fluorescent protein (GFP) fusion gene were generated and tested for growth and stress-responsive gene expression under mannitol and NaCl-stressed conditions. A construct with PgWRKY74 enabled yeast reporter cells to survive on test media in the yeast one-hybrid assays. The electrophoretic mobility of DNA with putative WRKY TF-binding motifs was lower in the presence of a recombinant PgWRKY74 protein than its absence. The PgWRKY74-GFP-overexpressing Arabidopsis plants exhibited smaller rosette areas than did wild-type plants under mannitol-stressed and NaCl-stressed conditions, and exhibited weaker expression of RD29B, which is induced by the stress-related phytohormone abscisic acid (ABA), under the mannitol-stressed condition. PgWRKY74 have transcriptional activation potential and DNA-binding ability, and can negatively regulate plant responses to mannitol and NaCl stresses, possibly by decreasing ABA levels or ABA sensitivity.

Abstract 2914: AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation

Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2914.

Efficacy of cartilage-targeted IGF-1 in a mouse model of growth hormone insensitivity.

Recombinant human IGF-1 is used to treat severe primary IGF-1 deficiency, but this treatment requires twice-daily injection, often does not fully correct the growth deficit, and has important off-target effects. We therefore sought to target IGF-1 to growth plate cartilage by generating fusion proteins combining IGF-1 with single-chain human antibody fragments that target matrilin-3, a cartilage matrix protein. We previously showed that this cartilage-targeting IGF-1 fusion protein (CV1574-1) promoted growth plate function in a GH-deficient (lit) mouse model. Here, we studied CV1574-1 in a second mouse model, C57BL/6 wild-type mice treated with pegvisomant to induce GH resistance. In this model, once-daily injections of CV1574-1 for 5 days partially restored the pegvisomant-induced decrease in growth plate height without increasing kidney cell proliferation. Furthermore, we found that subcutaneous CV1574-1 showed significantly reduced hypoglycemic effect compared to injection of IGF-1 itself. Lastly, to gain mechanistic insights into the role of matrilin-3 targeting, we assessed the ability of CV1574-1 to activate AKT signaling in vitro and found that CV1574-1 caused a prolonged increase in AKT signaling compared to IGF-1 and that this effect was dependent on matrilin-3. Taken together, our findings provide further evidence that cartilage-targeted therapy could provide new pharmacological approaches for the treatment of childhood growth disorders, such as GH-insensitivity syndrome.

Generation and Characterization of ORF55/ORF57-Deleted Recombinant Cyprinid herpesvirus 2 Mutants with Chimeric Capsid Protein Gene of Grouper Nervous Necrosis Virus.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen that causes significant losses to the global aquaculture industry due to mass mortality in crucian carp and goldfish. This study demonstrates that the ORF55/ORF57 deletion mutants CyHV-2-Δ55-CP and CyHV-2-Δ57-CP obtained through homologous recombination replicate effectively within the caudal fin of Carassius auratus gibelio (GiCF) cells and exhibit morphologies similar to the CyHV-2 wild-type strain. Both mutants demonstrated a decrease in virulence, with CyHV-2-Δ57-CP exhibiting a more significant reduction. This serves as a reference for the subsequent development of recombinant attenuated vaccines against CyHV-2. Additionally, both mutants expressed the inserted RGNNV-CP (capsid protein of Redspotted grouper nervous necrosis virus) fusion protein gene, and inoculation with CyHV-2-Δ57-CP-infected GiCF cell lysates elicited an antibody response in the grouper. These results indicate that, while ORF55 and ORF57 genes of CyHV-2 are not required for viral replication in vitro, they do play a role in virulence in vivo. Additionally, expression of foreign protein in CyHV-2 suggests that the fully attenuated mutant of CyHV-2 could potentially function as a viral vector for developing subunit vaccines or multivalent recombinant attenuated vaccines.

Targeted expression of a designed fusion protein containing BMP2 into the lumen of exosomes

BackgroundExosomes are 30–150 nm membrane vesicles, originating from the endocytic pathway. By acting as natural carriers of biomolecules, they can transfer various materials to recipient cells. Therefore, discovering novel strategies for cargo packaging into exosomes is crucial. MethodsThe fusion constructs, consisting of protein of interest (BMP2) along with the targeting motif, linkers, tracking proteins, and enzyme cleavage sites, were computationally designed. Following the homology modeling, the best structure was selected and subjected to molecular dynamics (MD) simulation and docking analyses. The fusion protein gene was expressed in the HEK-293LTV cell line. The high-efficiency transfected and transduced cells were screened and their exosomes were isolated. Finally, cell and exosome lysates were evaluated for expression of the fusion protein. ResultsA total of 12 constructs with lengths ranging from 483 to 496 were designed. The top three templates, 1REW, 2H5Q, and 2MOF were screened. MD simulation and docking analyses of the structures revealed their stability and functionality. In the protein expression analyses, three bands at sizes of approximately 60, 25, and 12.5 kDa were observed, consistent with the sizes of the complete fusion protein, dimeric, and monomeric BMP2 protein. The presence of a 12.5 kDa band at exosome lysate analysis might suggest that it was loaded and cleaved inside exosomes. ConclusionIn summary, these findings revealed that the proposed idea for cargo sorting within the exosome lumen through incorporating an appropriate cleavage site was effective, thus providing further insight into the potential of exosomes as nano-shuttles bearing therapeutic biomolecules.

Serological Detection, Isolation and Molecular Confirmation of Parainfluenza Virus-3 in Camels, Iraq

The objectives of this study were to detect and isolate the Parainfluenza-3 virus (PIV-3) in camels with naturally developed respiratory illness and to determine the titer of the isolates using the virus titration. Therefore, an overall 100 nasal swabs and jugular vein blood samples were collected from diseased camels in four districts in Wasit province (Iraq) from December (2019) to March (2020). The swabs were subjected to six subsequent passages on bovine kidney cell culture (BKCC) to isolate the virus and to confirm infection by molecular PCR assay. Fever (40°C), abundant runny nasal discharge, ocular discharge, coughing, depression, increased respiratory rate, abnormal breath sounds, and mainly wheezing are the most observed clinical signs. Positive findings were involved 24% by ELISA and 37% by RT-PCR. The age group from 1-2 years old showed a high infection rate, while the lower level was in the 4-6 years old group. Regarding season, the infection rate was high in winter compared to spring. Sheik Saad city appeared to have a higher infection rate than other districts. The positive samples inoculated into the Bovine kidney cell culture (BKCC) revealed the cytopathic effects (CPE) after three successive passages, which appeared as clumping and rounding with the progression of infection time at the 4th passage. Elongation and giant cell formation were shown in some isolates after the 5th and 6th passages until they reached complete detachments of the cells from the cell sheet. The titer of viral tissue culture infective dose (TCID50) of the 3rd passage was determined in BKCC cells at 10–3/0.05 ml, and the high titer was shown at the 5th and the 6th passages equal to 10-5/ 0.05 ml. In conclusion, PIV-3 is widespread among camels infected with respiratory illness; therefore, studies are necessary to detect the prevalence rate among camels in other Iraqi regions. Keywords: PIV-3, Fusion protein gene, Hemagglutination protein gene, ELISA, PCR

Single-dose mucosal replicon-particle vaccine protects against lethal Nipah virus infection up to 3 days after vaccination.

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

Epidemiological and genetic analysis of Cetacean Morbillivirus circulating on the Italian coast between 2018 and 2021.

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea.

MicroRNAs Involved in Nutritional Regulation During Plant-Microbe Symbiotic and Pathogenic Interactions with Rice as a Model.

Plants are constantly challenged with numerous adverse environmental conditions, including biotic and abiotic stresses. Coordinated regulation of plant responses requires crosstalk between regulatory pathways initiated by different external cues. Stress induced by excessiveness or deficiency of nutrients has been shown to positively or negatively interact with pathogen-induced immune responses. Also, colonization by arbuscular mycorrhizal (AM) fungi can improve plant nutrition, mainly phosphorus and resistance to pathogen infection. The proposed review addresses these issues about a new question that integrates adaptation to nutrient stress and disease resistance. The main goal of the current review is to provide insights into the interconnected regulation between nutrient signaling and immune signaling pathways in rice, focusing on phosphate, potassium and iron signaling. The underpinnings of plant/pathogen/AM fungus interaction concerning rice/M. oryzae/R. irregularis is highlighted. The role of microRNAs (miRNAs) involved in Pi (miR399, miR827) and Fe (miR7695) homeostasis in pathogenic/symbiotic interactions in rice is discussed. The intracellular dynamics of membrane proteins that function in nutrient transport transgenic rice lines expressing fluorescent protein fusion genes are outlined. Integrating functional genomic, nutritional and metal content, molecular and cell biology approaches to understand how disease resistance is regulated by nutrient status leading to novel concepts in fundamental processes underlying plant disease resistance will help to devise novel strategies for crop protection with less input of pesticides and fertilizers.