Nucleotide Sequence Research Articles

HTLV infection in urban population from Mato Grosso do Sul, Central Brazil.

Brazil has the highest number of HTLV-1 infection in Latin America, with around one million cases spread unevenly across regions. However, there is a limited number of studies on this infection in the general population. This cross-sectional study aimed to estimate the prevalence of HTLV as well as identify types, and subtypes of HTLV among the urban population of Campo Grande, capital of Mato Grosso do Sul state (MS). Between July 2023 and March 2024, all information was obtained from self-reported interviews, and blood samples were collected and screened for anti-HTLV-1/2 by immunoassay and confirmed using the immunoblot method. The proviral DNA of HTLV-1/2 in positive samples was quantified by real-time PCR (qPCR) and genotyped by nucleotide sequencing (Sanger's method). The study enrolled 611 participants, with the majority being women (90.54%), mixed race (46.32%), heterosexual (87.64%), and with a median age of 39years. The prevalence rate of anti-HTLV-1 infection was 0.82% (CI 95% 0.34-1.96). All positive samples (n = 5) were identified as belonging to the Cosmopolitan subtype, one belonging to Japanese and four to Transcontinental subgroups. Among the five positive individuals, two presented symptoms associated with HTLV-1 infection. This study highlights an intermediate prevalence of HTLV-1 in the urban population of Campo Grande, Mato Grosso do Sul, and provides epidemiological information that could help bridge the gaps in the distribution of HTLV in the general population. Also, medical care was provided for individuals presenting clinical manifestations who were previously unaware of their serological status.

Unveiling the dynamics of hepatitisC virus transmission among injection drug users and men who have sex with men: A comprehensive study in Japan.

In Japan, despite low nationwide hepatitisC (HCV) incidence, new infections among people who inject drugs (PWID) and men who have sex with men (MSM) hinder HCV elimination. We explored HCV transmission dynamics and screened HCV recombination within these populations. This cross-sectional study recruited HCV-infected patients from Osaka National Hospital, Osaka, Japan, from January 2010 to September 2023. Data from questionnaires and medical records were analyzed. Serum samples collected before anti-HCV treatment underwent HCV RNA extraction, and sequencing of full core (576bp) and NS5B (267bp) regions using the Sanger method. Genotype distribution was determined by phylogenetic analysis, and recombinant screening was conducted. A total of 115 patients were categorized into non-MSM PWID (31), MSM PWID (15), MSM non-PWID (25), and non-MSM non-PWID (44). Positive amplification rates were 99.1% (114/115) for the full-core region, and 96.5% (111/115) for NS5B. No intergenotypic recombination was detected. The predominant genotype in non-MSM PWID was 2a (58%), whereas genotype 1b was most common in MSM PWID, MSM non-PWID, and non-MSM non-PWID groups (79%, 64%, and 68%, respectively). Nucleotide sequence similarity of 94.75%-100% was found in HCV strains from MSM PWID and MSM non-PWID in both full-core and NS5B regions, whereas strains from non-MSM PWID and non-MSM non-PWID were distinct. The findings suggest that the transmission route in PWID is determined by MSM status, whereas MSM groups showed the same transmission route regardless of PWID. HCV control measures should be focused not only on PWID, but also on MSM to achieve HCV elimination in Japan.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Systematics of Pseudocavisoma (Acanthocephala: Echinorhynchida): assessment of familial affiliation, establishment of a new species, and complementary redescription of the type species based on syntypes.

The familial affiliation of the so-far-monotypic palaeacanthocephalan genus Pseudocavisoma Golvan & Houin, 1964 has solely been based on morphological characteristics, lacking nucleotide sequence data. In this paper, we assess the phylogenetic position of the genus with a dataset consisting of partial sequences of the mitochondrial cytochrome c oxidase subunit I, and the nuclear 18S and 28S rRNA genes for 37 species of palaeacanthocephalans available in public databases along with the ones determined from the new species Pseudocavisoma setoensesp. nov., herein established with specimens collected from the intestine of the pearl-spot chromis Chromis notata (Temminck & Schlegel, 1843) obtained in the Seto Inland Sea, Japan. Our results suggested that Pseudocavisoma should be placed in Micracanthorhynchinidae Yamaguti, 1963, rather than in Cavisomatidae Meyer, 1932 as had been considered by previous authors. Because Micracanthorhynchinidae has been regarded synonymous with Rhadinorhynchidae Lühe, 1912 since 1985, we re-validate the former taxon while providing an amended diagnosis for it. Pseudocavisoma setoense sp. nov. can be distinguished from the sole congener Pseudocavisoma chromitidis (Cable & Quick, 1954) by the distributional pattern and the size of spines on the trunk. We provide a partial morphological redescription of the latter species based on syntypes of Cavisoma chromitidis Cable & Quick, 1954. We also provide an amended diagnosis for Pseudocavisoma to accommodate P. setoense sp. nov.

No evidence that mutations in SARS-CoV-2 variants of concern derive from homologous fragments in gut microbiota.

Understanding the origin and evolution of mutations in SARS-CoV-2 variants of concern (VOCs) is a critical area of research. B. Cao, X. Wang, W. Yin, Z. Gao, and B. Xia (mBio 15:e03187-23, 2024, https://doi.org/10.1128/mbio.03187-23) proposed that these mutations originated from bacterial sequences incorporated into the viral genome through stochastic template-switching by the viral RNA-dependent RNA polymerase (RdRp). Their analysis suggested that 62% of the viral mutation fragments (VMFs) in key SARS-CoV-2 proteins were identical to bacterial protein sequences. Given the implications of this finding, we re-examined the methods employed and argue that they resulted in false-positive findings. Specifically, the short query length of VMFs, seven amino acids, leads to spurious matches in large protein databases, as indicated by high BLAST Expect values. Furthermore, we analyzed the nucleotide sequence of VMFs, revealing no unique homology between SARS-CoV-2 and bacterial sequences. Consequently, the evidence does not support the hypothesis that bacterial sequences contribute to the evolution of SARS-CoV-2 VOCs. Instead, the emergence of these variants is more plausibly attributed to factors intrinsic to viral replication and evolution, such as the error-prone nature of RdRp, intrahost diversity, and recombination of related viral sublineages.

Population Genetic Dissection of HLA-DPB1 Amino Acid Polymorphism to Infer Selection

Although allele frequency data for most HLA loci provide strong evidence for balancing selection at the allele level, the DPB1 locus is a notable exception, with allele frequencies compatible with neutral evolution (genetic drift) or directional selection in most populations. This discrepancy is especially interesting as evidence for balancing selection has been seen at the nucleotide and amino acid (AA) sequence levels for DPB1. We describe methods used to examine the global distribution of DPB1 alleles and their constituent AA sequences. These methods allow investigation of the influence of natural selection in shaping DPβ diversity in a hierarchical fashion for DPB1 alleles, all polymorphic DPB1 exon 2-encoded AA positions, as well as all pairs and trios of these AA positions. In addition, we describe how asymmetric linkage disequilibrium for all DPB1 exon 2-encoded AA pairs can be used to complement other methods. Application of these methods provides strong evidence for the operation of balancing selection on AA positions 56, 85–87, 36, 55 and 84 (listed in decreasing order of the strength of selection), but no evidence for balancing selection on DPB1 alleles.

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

Genetic profile of carbapenem-resistant <i>Acinetobacter baumannii</i> strains

The purpose of the research: the molecular genetic characteristic ofA. baumanniicarbapenem-resistant strains, including clonal affiliation, resistome and virulome pattern analysis, description of genetic environment resistance determinants, comparative genetic analysis, and assessment of phylogenetic relationships are presented in the work. The studiedA. baumanniistrains belonged to sequence types ST2Pas/ST2062.2063Oxf(n = 5) and ST78Pas/ST1757Oxf(n = 2). The nucleotide sequences of the studiedA. baumanniiST2Pas/ST2062.2063Oxfstrains were grouped into a single cluster according to phylogenetic analysis. And the sequences of theA. baumanniiST78Pas/ST1757Oxfstrains were combined with the nucleotide sequence of theA. baumanniiAbCTX5 strain, isolated in France in 2015. The presence of intrinsic (OXA-51-like) and acquired carbapenemases genes was shown inA. baumanniistrains. In particular,blaOXA-23are identified in members of ST2Pas/ST2062.2063Oxf, and inA. baumanniiST78Pas/ST1757Oxfstrains —blaOXA-72as part of the plasmid DNA. TheA. baumanniiST78Passtrains possessed additional extended-spectrum beta-lactamase genes. Тhe CTX-M-115 cephalosporinase gene are present in both strains, and theA. baumanniistrain NNAb_2023.3 has theblaCARB-16gene. MostA. baumanniistrains are characterized by the presence of acquired genes for enzymatic modification of macrolides(mph(E), msr(E)), chloramphenicols(catB8), aminoglycosides(aadA, aph(3'')-Ib, aph(6)-Id, aac(6')-Ib, aph(3')-Ia, armA). A comparative analysis showed that inA. baumanniiST2Pas/ST2062.2063Oxfstrains the resistance determinants to macrolides and aminoglycosides are located in the Tn6279-like transposon, and the aminoglycosidases genesaph(3'')-Ib, aph(6)-Idare associated with theIS91-like mobile element. InA. baumanniiST78Pas/ST1757Oxfstrains, resistance genes to aminoglycosides, macrolides, sulfonamides, and beta-lactamase genes are grouped in a region from 60 to 80 Kb long between theglmSandhutCgenes. The presence of mutations in thegyrAandparCgenes associated with resistance to fluoroquinolones were characterized in allA. baumanniistrains. Thus, new knowledge about the genetic profile of carbapenem-resistantA. baumanniistrains representing epidemically significant international clonal lineage has been obtained.

Proteus Syndrome: Description of Two Clinical Cases

Background. Proteus syndrome is extremely rare congenital multisystem disease with high variability in clinical manifestations. Its prevalence is unknown, there are less than 200 cases in the world literature. The syndrome is a classic example of somatic mosaicism, and all target drugs for its management are based on it. Clinical case description. This article describes two clinical cases with somatic variants of the nucleotide sequence in the AKT1 gene, mosaic form, revealed by the NGS method. Target drug (mTOR-inhibitors group) was assigned in one case. Conclusion. The description of the phenotypic features of patients with Proteus syndrome is crucial as this pathology is very rare. It is necessary to increase the awareness of clinicians about this disease to develop a plan for dynamic follow-up with consideration to life-threatening complications (malignant tumors and thrombembolia risk). Genetic verification of Proteus syndrome is mandatory nowadays as target therapy is actively developed and implemented, thus, revision of clinical guidelines is recommended.

Molecular phylogenetic and biogeographic evidence of Lepidagathis Willd. (Acanthaceae, Barlerieae) focusing on Indian endemics

Lepidagathis Willd., a genus belonging to the Acanthaceae family, is primarily distributed in tropical and subtropical regions worldwide, encompassing approximately 153 species. While considerable morphological research has been conducted on Lepidagathis, it has not completely dispelled taxonomic ambiguities and conflicting interpretations. Molecular analysis emerges as a valuable tool for resolving these taxonomic uncertainties, but the availability of nucleotide sequence data for Lepidagathis has been limited thus far. This study delivers a phylogenetic analysis of Lepidagathis species, utilizing both chloroplast and nuclear regions. The results of Bayesian Inference and Maximum Likelihood phylogenetic analyses consistently segregate the studied Lepidagathis species into two principal clades, denoted as Clade A and Clade B. Notably, this analysis firmly positions the Indian endemic Lepidagathis within Clade A, supported by robust statistical evidence. Furthermore, our biogeographical analysis strongly suggests that the origin of Lepidagathis might be traced back to Eurasia. This research establishes a foundational molecular phylogeny of Lepidagathis, offering valuable insights for future taxonomic investigations. Additionally, it sheds light on the evolutionary history and biogeographical origins of the Lepidagathis genus.

Genetic Spatio-Temporal Analysis of Hepatitis D Virus Infection in Western Brazilian Amazon

The Brazilian Amazon region is a highly endemic area for hepatitis Delta and has areas that are difficult to access. Understanding the dynamics of HDV transmission in these remote locations is important for elucidating the routes of infection. To investigate this, a molecular analysis of HDV was conducted to assess the spatio-temporal dynamics of HDV cases. Between 2010 and 2023, 35 patients were recruited from the Viral Hepatitis Outpatient Clinic in Rondônia, Brazil. Conventional PCR was used to amplify the complete HDV genome followed by nucleotide sequencing via the Sanger method. The HDV genotype was determined using maximum likelihood phylogenetic reconstruction. A Skygrid coalescent approach with a Relaxed Random Walk phylogeographic model was used for the spatio-temporal analysis. Most individuals were males (21/35), with a median age of 39 years. HDV-3 was identified in all samples (35/35; 100%). The tMRCA was estimated to be 1824, with a substitution rate of 8.2 × 10−4 substitutions/site/year. The results suggest that HDV likely entered Brazil around 1820, in the state of Amazonas, subsequently spreading to Acre and Rondônia. Notable migration events were observed starting from 2010. This study suggests that HDV-3 has a complex evolutionary history spanning over two centuries, with intricate transmission routes in different locations in Brazil.

Survey of sand fly fauna in six provinces of Southern Vietnam with species identification using DNA barcoding

BackgroundSand flies, belonging to the Psychodidae family, represent small, hairy insects that serve as significant vectors in various important medical and veterinary diseases. Despite being recognized by the World Health Organization as an endemic area for leishmaniasis, Southeast Asia lacks comprehensive information on the species composition and biology of sand flies. To address this, the current study aimed to survey sand fly biodiversity.MethodsSand flies from six provinces in Southern Vietnam were collected using CDC light traps. Sand flies were subsequently identified morphologically and confirmed molecularly using mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (cytb) sequences. BLASTN searches were conducted, and the species identity of sand flies was further confirmed through a Barcode of Life Database (BOLD) search utilizing COI sequences. Subsequently, nucleotide sequences were subjected to a panel of analyses including intraspecific variation, phylogenetic relationships and haplotype network. The average densities of collected sand flies (sand flies/trap/night) and species richness were also recorded.ResultsA total of 753 sand flies were collected. After excluding damaged specimens, six sand fly species, namely Phlebotomus stantoni, Sergentomyia khawi, Se. silvatica, Se. barraudi, Se. bailyi and Grassomyia indica, were identified. All conspecific sand fly sequences, including Ph. stantoni, Se. barraudi, Gr. indica, Se. bailyi, Se. khawi and Se. silvatica, clustered with their reference sequences, corroborating the results of morphology-based identification, BLASTN analysis and BOLD search. For intraspecific variation of sand flies obtained from the current study, COI diversity indices were consistently higher than those of cytb.ConclusionsThis study provides the first updates on morphological and molecular characterization of sand flies in Southern Vietnam. This acquired knowledge on sand fly species composition is essential for controlling sand fly-borne diseases in this potentially endemic region.Graphical

Link Between Individual Codon Frequencies and Protein Expression: Going Beyond Codon Adaptation Index

An important role of a particular synonymous codon composition of a gene in its expression level is well known. There are a number of algorithms optimizing codon usage of recombinant genes to maximize their expression in host cells. Nevertheless, the underlying mechanism remains unsolved and is of significant relevance. In the realm of modern biotechnology, directing protein production to a specific level is crucial for metabolic engineering, genome rewriting and a growing number of other applications. In this study, we propose two new simple statistical and empirical methods for predicting the protein expression level from the nucleotide sequence of the corresponding gene: Codon Expression Index Score (CEIS) and Codon Productivity Score (CPS). Both of these methods are based on the influence of each individual codon in the gene on the overall expression level of the encoded protein and the frequencies of isoacceptors in the species. Our predictions achieve a correlation level of up to r = 0.7 with experimentally measured quantitative proteome data of Escherichia coli, which is superior to any previously proposed methods. Our work helps understand how codons determine protein abundances. Based on these methods, it is possible to design proteins optimized for expression in a particular organism.

APPLICATION OF THE DNA BARCODING METHOD FOR IDENTIFICATION AND TAXONOMIC ANALYSIS OF THE LAMIACEAE FAMILY

The Lamiaceae family is one of the largest and most important among the aromatic plants, including many species with pronounced pharmacological properties. Plants of this family, such as mint, rosemary and thyme, are widely used in traditional and modern medicine due to their therapeutic and biochemical properties. The importance of correct species identification is emphasized by the need to preserve the biodiversity and sustainable use of the resources of this family. In this work, plants of Lamiaceae family growing in the territory of the Karaganda region were studied by DNA barcoding. The studies were conducted on the basis of nucleotide sequences of rbcL and trnH-psbA chloroplast DNA barcodes using the methods of paired genetic distances, BLASTn and phylogenetic tree based on the analysis of maximum parsimony. The high efficiency of the rbcL locus was demonstrated for the identification of higher taxonomic groups, such as subfamilies and tribes, while the intergenic spacer trnH-psbAhasproved to be effectiveat the level of speciesandgenus level.

Morphological and molecular evidence for a new species of Russula subgen. Compactae from Pakistan

A new species of Russula has been collected from Punjab and Khyber pakhtunkhwa provinces, Pakistan. Russula punjabensis sp. nov. is characterized by grayish purple to grayish red pileus with brittle central region, white lamellae with brown striations, becoming brown at maturity, a rhizoidal base, small (7.48 × 6.64 µm) subglobose to broadly ellipsoid basidiospores as compared to R. acrifolia (7.7 µm), small (34.80 × 4.35 µm) cylindrical to narrowly fusiform pileocystidia and presence of clamp connections in pileipellis. Phylogenetic analysis based on nuclear ribosomal ITS & LSU nucleotide sequences confirm its novelty.

TargetCall: eliminating the wasted computation in basecalling via pre-basecalling filtering

Basecalling is an essential step in nanopore sequencing analysis where the raw signals of nanopore sequencers are converted into nucleotide sequences, that is, reads. State-of-the-art basecallers use complex deep learning models to achieve high basecalling accuracy. This makes basecalling computationally inefficient and memory-hungry, bottlenecking the entire genome analysis pipeline. However, for many applications, most reads do not match the reference genome of interest (i.e., target reference) and thus are discarded in later steps in the genomics pipeline, wasting the basecalling computation. To overcome this issue, we propose TargetCall, the first pre-basecalling filter to eliminate the wasted computation in basecalling. TargetCall’s key idea is to discard reads that will not match the target reference (i.e., off-target reads) prior to basecalling. TargetCall consists of two main components: (1) LightCall, a lightweight neural network basecaller that produces noisy reads, and (2) Similarity Check, which labels each of these noisy reads as on-target or off-target by matching them to the target reference. Our thorough experimental evaluations show that TargetCall 1) improves the end-to-end basecalling runtime performance of the state-of-the-art basecaller by 3.31× while maintaining high (98.88%) recall in keeping on-target reads, 2) maintains high accuracy in downstream analysis, and 3) achieves better runtime performance, throughput, recall, precision, and generality than prior works. TargetCall is available at https://github.com/CMU-SAFARI/TargetCall.

First detection of D181 genotype of infectious bronchitis in poultry flocks of Morocco

BackgroundThis paper reports the first pathological and molecular characterization of the novel variant of infectious bronchitis virus (IBV) D181 in poultry flocks in Morocco and Africa.MethodsThe study includes six poultry farms, involving three flocks of layers aged between 28 and 67 weeks and three broiler flocks aged 27, 39 and 42 days from different regions of Morocco. In all affected layer flocks, a severe drop in egg production with poor eggshell quality was reported. Necropsy of dead birds was carried out, and samples of trachea, lungs, oviduct, ovaries, and kidneys were fixed in 10% neutral buffered formalin for histopathologic examinations, while other portions were stored at -20 °C for molecular analysis. Real time RT-qPCR for IBV gene group was performed, and IBV variants were identified. Partial S1 gene sequences were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic and amino acid similarity analysis.ResultsNecropsy of dead birds revealed misshapen and hemorrhagic ovarian follicles with an edematous oviduct and severe reaction in the cecal tonsils. A caseous material accumulation in the sinus was noted in few birds. In contrast, the broiler flocks exhibited respiratory clinical signs such as difficulty in breathing, sneezing, tracheal rales, watery eyes and lethargy, associated with a decrease in feed consumption. Mortality in broiler ranged from 2 to 15%. Histopathological analysis of samples showed a lympho-plasmocytic inflammation in the oviduct, trachea, and lungs. Individual necrosis of epithelial cells, with sloughing of the bronchial epithelium and accumulation of desquamated cells with mucus in the airways, was observed in some birds. Partial S1 gene sequencing and phylogenetic analyses showed that the Moroccan strains were very closely related to D181 strains isolated in Dutch layers and breeders in 2018. Nucleotide sequence identities reached 90.9–95% with the Dutch isolates (strain CK/NL/D181/2018).ConclusionOur sequencing results demonstrate for the first time that the D181 IBV genotype is circulating in Moroccan poultry. These findings justify permanent monitoring of circulating strains in order to appropriately adjust vaccination strategies to align with the evolving field situation.

Molecular Detection and Isolation of Chicken Astrovirus from Commercial Chicken Flocks of Kerala, India

Reduced productivity due to enteric infections in chickens is a very common challenge in the poultry industry of India. Various studies associated Chicken astrovirus (CAstV) as a common pathogen in commercial flocks exhibiting poor weight gain, stunted growth and visceral gout. However, no published report documented the detection of the virus from Kerala so far. Hence, this study was done to investigate the presence of CAstV in 50 broiler flocks from different parts of Kerala with clinical signs such as retarded growth, diarrhoea, and a mortality of 5 to 10 %. Out of the 50, five flocks were found positive for the virus by using the Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) method with the primers targeting the partial ORF1b gene of the viral nucleic acid. The viral presence was confirmed by isolation using embryonated chicken eggs (ECE), which produced notable alteration to the infected embryos. Nucleotide sequencing of the isolates revealed a high identity of up to 100 % with the other CAstV sequences retrieved from the NCBI database. The study confirms the first report of molecular detection and isolation of CAstV from the state of Kerala.

Novel dual-pathogen multi-epitope mRNA vaccine development for Brucella melitensis and Mycobacterium tuberculosis in silico approach.

Brucellosis and Tuberculosis, both of which are contagious diseases, have presented significant challenges to global public health security in recent years. Delayed treatment can exacerbate the conditions, jeopardizing patient lives. Currently, no vaccine has been approved to prevent these two diseases simultaneously. In contrast to traditional vaccines, mRNA vaccines offer advantages such as high efficacy, rapid development, and low cost, and their applications are gradually expanding. This study aims to develop multi-epitope mRNA vaccines argeting Brucella melitensis and Mycobacterium tuberculosis H37Rv (L4 strain) utilizing immunoinformatics approaches. The proteins Omp25, Omp31, MPT70, and MPT83 from the specified bacteria were selected to identify the predominant T- and B-cell epitopes for immunological analysis. Following a comprehensive evaluation, a vaccine was developed using helper T lymphocyte epitopes, cytotoxic T lymphocyte epitopes, linear B-cell epitopes, and conformational B-cell epitopes. It has been demonstrated that multi-epitope mRNA vaccines exhibit increased antigenicity, non-allergenicity, solubility, and high stability. The findings from molecular docking and molecular dynamics simulation revealed a robust and enduring binding affinity between multi-epitope peptides mRNA vaccines and TLR4. Ultimately, Subsequently, following the optimization of the nucleotide sequence, the codon adaptation index was calculated to be 1.0, along with an average GC content of 54.01%. This indicates that the multi-epitope mRNA vaccines exhibit potential for efficient expression within the Escherichia coli(E. coli) host. Analysis through immune modeling indicates that following administration of the vaccine, there may be variation in immunecell populations associated with both innate and adaptive immune reactions. These types encompass helper T lymphocytes (HTL), cytotoxic T lymphocytes (CTL), regulatory T lymphocytes, natural killer cells, dendritic cells and various immune cell subsets. In summary, the results suggest that the newly created multi-epitope mRNA vaccine exhibits favorable attributes, offering novel insights and a conceptual foundation for potential progress in vaccine development.

Immuno-RCA for highly sensitive detection of the antigen-antibody complex in the blood group antigen model

The problem of detecting tiny quantities of analytes by immunochemical methods has been tried for a long time. One approach is to use nucleic acid amplification methods to amplify the signal from a single antigen-antibody interaction. An amplification method suitable for microarrays is the rolling circle amplification reaction. The principle of the method is usage of a conjugate of a detecting antibody with a primer and subsequent isothermal amplification. The generation of a huge single-stranded reaction product starts after adding the necessary components for amplification reaction: circular oligonucleotides, which serves as a template for amplification and phage phi29 polymerase with the other components. This reaction product consists of a lot of repeats of a nucleotide sequence, that is complementary to the circular template. The fluorescent DNA probe can hybridize to each repeat on the product molecule, resulting in a significantly higher level of fluorescence than with fluorescently labeled antibody or streptavidin development systems. In addition, the reaction product remains immobilized on the surface, allowing usage of this approach for the detection of antigen-antibody interactions in other solid-phase analysis systems, such as microarrays. A common problem with such approaches is the nonspecific sorption of components of the immunochemical reaction or amplification reaction, leading to a high background. It is obvious that no matter how highly sensitive the analysis is in theory, a high background will reduce the entire potential of the method to nothing. Herein, we have developed a method that makes it possible to detect small amounts of antibodies to glycans in blood serum and in swabs from tumor cells in a microarray format using a model of blood group antigens. It was possible to obtain a 30 to 70-fold increase of fluorescence level from a specific interaction compared to the use of fluorescently labeled streptavidin. The method we are developing is promising, as it allows us to significantly increase the signal from the specific antigen/antibody interaction in the glycochip format, which will make it possible to detect antibodies to glycans in samples with a very low concentrations of antibodies, for example, in washes from tumor cells.