Inferring B-cell derived T-cell receptor induced multi-epitope-based vaccine candidate against enterovirus 71: a reverse vaccinology approach.

Purpose: Enterovirus 71, a pathogen that causes hand-foot and mouth disease (HFMD) is currently regarded as an increasing neurotropic virus in Asia and can cause severe complications in pediatric patients with blister-like sores or rashes on the hand, feet, and mouth. Notwithstanding the significant burden of the disease, no authorized vaccine is available. Previously identified attenuated and inactivated vaccines are worthless over time owing to changes in the viral genome. Materials and Methods: A novel vaccine construct using B-cell derived T-cell epitopes from the virulent polyprotein found the induction of possible immune response. In order to boost the immune system, a beta-defensin 1 preproprotein adjuvant with EAAAK linker was added at the N-terminal end of the vaccine sequence. Results: The immunogenicity of the designed, refined, and verified prospective three-dimensional-structure of the multi-epitope vaccine was found to be quite high, exhibiting non-allergenic and antigenic properties. The vaccine candidates bound to toll-like receptor 3 in a molecular docking analysis, and the efficacy of the potential vaccine to generate a strong immune response was assessed through in silico immunological simulation. Conclusion: Computational analysis has shown that the proposed multi-epitope vaccine is possibly safe for use in humans and can elicit an immune response.

Complete mitogenome sequence analysis of an Indian Black Bengal goat, Capra hircus, and its phylogenetic consequences.

Capra hircus (Black Bengal goat) is a prized goat breed reared across the Southeast Asian region. Known for its excellent chevon quality and adaptability to hot and humid climates, it supports the livelihood of millions of farmers. The present study reveals the complete mitogenome of a Black Bengal goat of Indian origin utilizing a next-generation sequencing approach (GenBank acc. no.-MZ073671). This newly assembled whole mitochondrial genome (16,637 bp) is composed of a non-coding control region (D-loop region), two ribosomal RNA (rRNA genes), 13 protein-coding genes, and 22 transfer RNA (tRNA). Overall, the current mitogenome shares 99.77% sequence homology with previously reported mitogenomes of Capra hircus. The phylogenetic analysis inferred that the current mitogenome shares a close relationship with the Teressa goat breed of the Andaman and Nicobar Islands.

Insights into genome plasticity and gene regulation in Orientia tsutsugamushi through genome-wide mining of microsatellite markers.

Microsatellite markers are being used for molecular identification and characterization as well as estimation of evolution patterns due to their highly polymorphic nature. The repeats hold 40% of the entire genome of Orientia tsutsugamushi (OT), but not yet been characterized. Thus, we investigated the genome-wide presence of microsatellites within nine complete genomes of OT and analyzed their distribution pattern, composition, and complexity. The in-silico study revealed that the genome of OT enriched with microsatellites having a total of 126,187 SSRs and 10,374 cSSRs throughout the genome, of which 70% and 30% are represented within the coding and non-coding regions, respectively. The relative density (RD) and relative abundance (RA) of SSRs were 42-44.43/kb and 6.25-6.59/kb, while for cSSRs this value ranged from 7.06 to 8.1/kb and 0.50 to 0.55/kb, respectively. However, RA and RD were weakly correlated with genome size and incidence of microsatellites. The mononucleotide repeats (54.55%) were prevalent over di- (33.22%), tri- (11.88%), tetra- (0.27%), penta- (0.02%), hexanucleotide (0.04%) repeats, with poly (A/T) richness over poly (G/C). The motif composition of cSSRs revealed that maximum cSSRs were made up of two microsatellites having unique duplication patterns such as AT-x-AT and CG-x-CG. To our knowledge, this is the first study of microsatellites in the OT genome, where characterization of such variations in repeat sequences would be important in deciphering the origin, rate of mutation, and role of repeat sequences in the genome. More numbers of microsatellites represented within the coding region provide an insight into the genome plasticity that may interfere with gene regulation to mitigate host-pathogen interaction and evolution of the species.

Activation of Host Cellular Signaling and Mechanism of Enterovirus 71 Viral Proteins Associated with Hand, Foot and Mouth Disease.

Enteroviruses are members of the Picornaviridae family consisting of human enterovirus groups A, B, C, and D as well as nonhuman enteroviruses. Human enterovirus type 71 (EV71) has emerged as a major cause of viral encephalitis, known as hand, foot, and mouth disease (HFMD), in children worldwide, especially in the Asia-Pacific region. EV71 and coxsackievirus A16 are the two viruses responsible for HFMD which are members of group A enteroviruses. The identified EV71 receptors provide useful information for understanding viral replication and tissue tropism. Host factors interact with the internal ribosome entry site (IRES) of EV71 to regulate viral translation. However, the specific molecular features of the respective viral genome that determine virulence remain unclear. Although a vaccine is currently approved, there is no effective therapy for treating EV71-infected patients. Therefore, understanding the host-pathogen interaction could provide knowledge in viral pathogenesis and further benefits to anti-viral therapy development. The aim of this study was to investigate the latest findings about the interaction of viral ligands with the host receptors as well as the activation of immunerelated signaling pathways for innate immunity and the involvement of different cytokines and chemokines during host-pathogen interaction. The study also examined the roles of viral proteins, mainly 2A and 3C protease, interferons production and their inhibitory effects.

Population genetic structure of fringe-lipped carp, Labeo fimbriatus from the peninsular rivers of India.

Labeo fimbriatus is a medium carp species found throughout India's peninsular river basins and is regarded as a valuable aquaculture resource alongside Indian major carps due to its taste and nutritional value. This species has recently declined dramatically due to habitat degradation and overfishing. Because of its enormous economic importance, a selective breeding programme is likely to be in place to improve performance traits. Knowledge of genetic variation among the base population from which the broodstock will be selected is an important step in this process. A diverse genetic base of broodstock is required to achieve the best response to selection for long-term aquaculture management practices. Consequently, using mitochondrial DNA (ATPase 6 and Control region) and microsatellite markers, we have made the first step toward estimating the level of genetic variation and how it is distributed among the four populations of L. fimbriatus found in peninsular rivers in India. The ATPase 6 gene analysis in four populations revealed 15 haplotypes and 51 variable sites, in contrast to the Control region, which had 60 haplotypes together with 73 variable sites and a haplotype diversity of 0.941. Twelve microsatellite loci displayed estimated allele numbers (N A) ranging from 3 to 19, observed heterozygosity (H O), and expected heterozygosity (H E), respectively, of 0.705 to 0.753 and 0.657 to 0.914. Each marker type showed a significant F ST value, indicating the presence of low to moderate genetic differentiation across entire wild populations. The Godavari, Kaveri, and Mahanadi populations formed one cluster according to the UPGMA, which was based on genetic distance matrix, while the Krishna population formed a separate cluster. The comparative genetic analysis of data from different markers utilized in the current study would enable the identification of the genetic stocks of L. fimbriatus and facilitate conservation measures and selective breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03369-y.

Gene expression and involvement of signaling pathways during host-pathogen interplay in Orientia tsutsugamushi infection.

Scrub typhus is a neglected tropical disease that affects one-third of the world's population. The disease is caused by Orientia tsutsugamushi (OT), an obligate intracellular Gram-negative bacterium. OT efficiently escapes from the endosomal pathway after entering the host cell and replicates inside cytosol. OT infection promotes cellular autophagy, the autonomous defense mechanism unlike other bacteria. This study has discussed the bacterial invasion process through the extracellular matrix and the immune response activated by the bacterium within the hosts. Furthermore, we have emphasized the importance of extracellular matrix and their cross-talk with the immune cells, such as, macrophages, neutrophils, and dendritic cells followed by their inflammatory response. We have also put an insight into the host factors associated with signaling pathways during scrub typhus disease with a special focus on the OT-induced stress response, autophagy, apoptosis, and innate immunity. Multiple cytokines and chemokines play a significant role in activating different immune-related signaling pathways. Due to the presence of high antigenic diversity among strains, the signaling pathways during the host-pathogen interplay of OT with its host is very complicated. Thus, it hinders to mitigate the severity of the pandemic occurred by the respective pathogen. Our investigation will provide a useful guide to better understand the virulence and physiology of this intracellular pathogen which will lead towards a better therapeutic diagnosis and vaccine development.

Recombination drives the emergence of orf virus diversity: evidence from the first complete genome sequence of an Indian orf virus isolate and comparative genomic analysis.

Contagious pustular dermatitis is a disease that primarily infects small ruminants and possesses zoonotic potential. It is caused by orf virus (ORFV), a member of the genus Parapoxvirus. In this study, we evaluated an ORFV outbreak in goats in Madhya Pradesh, a state in central India, during 2017. The transboundary potential of this virus was evaluated by constructing phylogenetic trees. The complete genome sequence of an ORFV isolate named Ind/MP/17 was found to be 139,807 bp in length with 63.7% GC content and 132 open reading frames (ORFs) flanked by 3,910-bp inverted terminal repeats (ITRs). An investigation into evolutionary parameters such as selection pressure (θ = dN/dS) and nucleotide diversity (π) demonstrated that ORFV has undergone purifying selection. A total of 40 recombination events were identified, 21 of which were evident in the Ind/MP/17 genome, indicating its ability to generate new variants.

Genome-wide identification and characterization of microsatellite markers within the Avipoxviruses.

Microsatellite markers or Simple Sequence Repeats (SSRs) are gaining importance for molecular characterization of the virus as well as estimation of evolution patterns due to its high-polymorphic nature. The Avipoxvirus is the causative agent of pox-like lesions in more than 300 birds and one of the major diseases for the extinction of endangered avian species. Therefore, we conducted a genome-wide analysis to decipher the type, distribution pattern of 14 complete genomes derived from the Avipoxvirus genus. The in-silico screening deciphered the existence of 917-2632 SSRs per strain. In the case of compound SSRs (cSSRs), the value was obtained 44-255 per genome. Our analysis indicates that the di-nucleotide repeats (52.74%) are the most abundant, followed by the mononucleotides (34.79), trinucleotides (11.57%), tetranucleotides (0.64%), pentanucleotides (0.12%) and hexanucleotides (0.15%) repeats. The specific parameters like Relative Abundance (RA) and Relative Density (RD) of microsatellites ranged within 5.5-8.12 and 33.08-53.58 bp/kb. The analysis of RA and RD value of compound microsatellites resulted between 0.25-0.82 and 4.64-15.12 bp/kb. The analysis of motif composition of cSSR revealed that most of the compound microsatellites were made up of two microsatellites, with some unique duplicated pattern of the motif like, (TA)-x-(TA), (TCA)-x-(TCA), etc. and self-complementary motifs, such as (TA)-x-(AT). Finally, we validated forty sets of compound microsatellite markers through an in-vitro approach utilizing clinical specimens and mapping the sequencing products with the database through comparative genomics approaches. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03169-4.

Molecular characterization and evolutionary analysis of Orientia tsutsugamushi in eastern Indian population.

Scrub typhus is a bacterial zoonotic acute febrile illness (AFI) caused by the obligate intracellular bacterium Orientia tsutsugamushi, which is an antigenically diverse strain frequently observed in the tropical region of Southeast Asian countries. The recent investigation was conducted to delineate the genotype identification of Orientia tsutsugamushi predominating in the eastern zone of India such as Odisha to decipher its strain type, and evaluate its diversity as well as evolutionary pattern based on the nucleotide analysis of the immune dominant 56 KDa gene. During this study, we have investigated 100 clinical samples (2014-2018), out of which 28 were positive for scrub typhus followed by its molecular characterization and phylogenetic analysis utilizing 56 KDa partial genes. Population genetic parameters showed the presence of 287 polymorphic sites within the analyzed 56 KDa gene. The gene diversity (Hd) and sequence diversity (π) was estimated 0.638 and 0.280, respectively. Selection pressure analysis (θ = dN/dS) having the value 0.222 suggests that the gene lied under purifying selection. The present study suggested a high rate of genetic diversity within the isolates. This research study sheds light on the hereditary and evolutionary relationships of Orientia strains found in the eastern Indian population. Understanding regional genetic variation is critical for vaccine development and sero-diagnostics methods. A significant level of genetic variability was observed during this study. This information has a way to understand more about antigen diversity that leads to develop an effective vaccine candidate for this pathogen.

Comparative analysis, distribution, and characterization of microsatellites in Orf virus genome.

Genome-wide in-silico identification of microsatellites or simple sequence repeats (SSRs) in the Orf virus (ORFV), the causative agent of contagious ecthyma has been carried out to investigate the type, distribution and its potential role in the genome evolution. We have investigated eleven ORFV strains, which resulted in the presence of 1,036-1,181 microsatellites per strain. The further screening revealed the presence of 83-107 compound SSRs (cSSRs) per genome. Our analysis indicates the dinucleotide (76.9%) repeats to be the most abundant, followed by trinucleotide (17.7%), mononucleotide (4.9%), tetranucleotide (0.4%) and hexanucleotide (0.2%) repeats. The Relative Abundance (RA) and Relative Density (RD) of these SSRs varied between 7.6-8.4 and 53.0-59.5 bp/kb, respectively. While in the case of cSSRs, the RA and RD ranged from 0.6-0.8 and 12.1-17.0 bp/kb, respectively. Regression analysis of all parameters like the incident of SSRs, RA, and RD significantly correlated with the GC content. But in a case of genome size, except incident SSRs, all other parameters were non-significantly correlated. Nearly all cSSRs were composed of two microsatellites, which showed no biasedness to a particular motif. Motif duplication pattern, such as, (C)-x-(C), (TG)-x-(TG), (AT)-x-(AT), (TC)- x-(TC) and self-complementary motifs, such as (GC)-x-(CG), (TC)-x-(AG), (GT)-x-(CA) and (TC)-x-(AG) were observed in the cSSRs. Finally, in-silico polymorphism was assessed, followed by in-vitro validation using PCR analysis and sequencing. The thirteen polymorphic SSR markers developed in this study were further characterized by mapping with the sequence present in the database. The results of the present study indicate that these SSRs could be a useful tool for identification, analysis of genetic diversity, and understanding the evolutionary status of the virus.

Identification and phylogenetic characterization of bovine ephemeral fever virus (BEFV) of Middle Eastern lineage associated with 2018-2019 outbreaks in India.

Bovine ephemeral fever virus (BEFV) is an evolving arbovirus reported across tropical, subtropical and temperate climatic zones globally. This study reveals prominent BEFV outbreaks in India, emerging annually during monsoons in subtropical areas accompanied by a congenial abundance of the vector population. PCR-based detection of viral genomic RNA in the blood samples collected during outbreaks of 2018-2019 for the first time confirmed the presence of BEFV in India. Phylogenetic analysis based on the glycoprotein gene of BEFV showed the current isolates to have high sequence homology with Middle Eastern lineage with nearly 97%, identity to Turkey (BEFV Ad12/TUR) and Israel (Israel 2006) isolates.

The emergence of subclades A1 and A3 avipoxviruses in India.

During the years 2010-2018, avipoxvirus (APV) outbreaks were observed in the domestic chickens and pigeons present in the eastern Indian state of Odisha. Based on typical pox lesions, followed by molecular techniques, the overall morbidity was found to be 18%-19.23% and 16.92%-23% in chickens and pigeons, respectively. The cutaneous forms of the disease were observed with varied rates of mortality, being 47.36%-52.77% in chickens and 39.13%-92% in pigeons. PCR amplification targeting the viral P4b core protein-coding gene and the DNA polymerase gene confirmed the presence of APV strains in 10 birds. Subsequent phylogenetic analysis of these two genes confirmed that the circulating strains were members of APV clade A. The subclade analysis revealed the introduction of A1 and A3 subclades in Indian chickens and pigeons, respectively. This study is the first molecular record of APVs circulating in eastern Indian birds (Odisha) and involves the first use of the polymerase gene to reveal the circulating clades of Indian APVs.