Molecular Evolutionary Relationships Research Articles

Molecular characterization and evolutionary relationships of DOFs in four cherry species and functional analysis in sweet cherry

The DOF (DNA binding with one finger) has multiple functions in plants. However, it has received little attention in the research field of cherries. In this study, the evolutionary relationship and molecular characterization of DOF in four cherry species were analyzed, revealing its expression pattern in sweet cherry. There are 23 members in Prunus avium cv. ‘Tieton’, 88 in Prunus cerasus, 53 in Cerasus × yedoensis, and 27 in Cerasus serrulata. Most of these genes are intron-less or non-intron, with a conserved C2-C2 domain. Due to heterozygosity and chromosomal ploidy, whole-genome duplication (WGD) events occur to varying degrees, and DOF genes are contracted during evolution. Furthermore, these genes are affected by purifying selection pressure. Under low-temperature treatment, the expression of PavDOF2 and PavDOF18 were significantly up-regulated, while that of PavDOF16 is significantly down-regulated. The expression of PavDOF9, PavDOF12, PavDOF14, PavDOF16, PavDOF17, PavDOF18, and PavDOF19 exhibits an increasing trend during flower development and varies during sweet cherry fruit development. PavDOF1, PavDOF8, PavDOF9, and PavDOF15 are localized in the nucleus but is not transcriptionally active. The findings systemically demonstrate the molecular characteristics of DOF in different cherry varieties, providing a basis for further research on the functions of these genes.

Population genetic diversity of tomato spotted wilt orthotospovirus isolates from tobacco in Yunnan Province, China

We explored the genetic diversity and molecular evolutionary characteristics of tomato spotted wilt orthotospovirus (TSWV) on tobacco in Yunnan. In this study, 523 diseased tobacco leaf samples with suspected TSWV infection from nine different geographical sources in Yunnan were tested, and nucleocapsid protein (NP) of some isolates was amplified and cloned by reverse transcription-polymerase chain reaction (RT‒PCR). Bioinformatics tools were used to analyze the population genetic structure and molecular evolutionary relationships of the tobacco TSWV population. TSWV was detected in 284 of 523 samples, with an average positivity rate of 54.3%. NP gene sequences of the 159 tobacco TSWV isolates obtained were all 777 bp, with 265 nucleic acid polymorphic sites, with 197 variant sites, including 66 parsimony informative and 131 single variant sites. Results for virus consistency and variation analysis based on the NP gene showed that the nucleotide sequence consistency of Yunnan TSWV isolates was 96∼100%, the haplotype diversity 0.963, and the nucleic acid diversity 0.006. Phylogenetic analysis revealed that Yunnan tobacco TSWV isolates belong to the same group, with lower genetic differentiation and more frequent gene exchange among tobacco isolates of different geographical origins; their geographical characteristics were not obvious, and significant recombination signals were detected in the gene sequences of the DL-20 and DL-42 isolates. Many of the NP gene mutations of tobacco TSWV isolates from different geographic origins were deleterious in the nine different planting regions of Yunnan tobacco TSWV isolates, with only the population in the QJ planting region experiencing past population expansion events. The findings suggest that genetic recombination, mutation, and negative selection pressure are major drivers of the genetic diversity of tobacco TSWV in Yunnan. This study report of genetic variation in populations of TSWV isolates from various tobacco regions in Yunnan and provides information on the population structure of Yunnan tobacco TSWV isolates, which can help to prevent and control TSWV on Yunnan tobacco and provide a reference for research of disease-resistant varieties of tobacco.

Molecular Characteristics and Evolutionary Relationships of Toll-Like Receptor (TLR21) of Indonesian KUB-1 Chicken

In vertebrates, the immune system has several Toll-like receptors (TLRs) to recognize specific pathogen-associated molecular patterns (PAMPs). In aves, TLR21 is known to be a functional homolog of TLR9 in mammals. This study aimed to determine the molecular characteristics of TLR21 in KUB-1 chicken, a superior breed of Indonesian local chicken, and their evolutionary relationship. The TLR21 gene was obtained from the ovarian RNA extraction and synthesized into cDNA by reverse transcription. We found that the cDNA sequence of the TLR21 of KUB-1 chicken has a length of 3504 bp, including a 2823 bp open reading frame (ORF), which encodes a putative protein of 940 amino acids (aa). The deduced KUB-1 chicken TLR21 protein consisted of 720 aa in the extracellular domain containing 20 LRRs, 23 aa in the transmembrane region, and 141 aa of Toll‐IL‐1 receptor in the intracellular domain, and had a molecular weight of 107 kDa. The KUB-1 chicken TLR21 had homology of about 99%, 76%, and 43% with other TLR21 from other breeds of chicken, non-chicken poultry, and fish, respectively. Its molecular character was conserved throughout the evolution of TLR21 in chicken, so the KUB-1 chicken was not much different from the broiler. However, there has been an evolution in duck and goose, although the function is still the same. Evolutionarily, Gallus gallus_KUB-1 TLR21, which belongs to the TLR11 Family, has differentiated from fish TLR21 and formed a paraphyletic relationship with TLR21 in Anser cygnoides and Anas platyrhynchos.

High throughput sequencing data reveals the complete mitogenome, abundance, maternal phylogeny, and mitogenomic diversity of Alectoris chukar of Iraq

Alectoris chukar is a wild game bird found in the north of Iraq, near the center of domestication and diversity of species. The mitogenome is one of the most vital resources for comprehensive studies of genetic diversity and molecular evolutionary relationships among avian species. In this study, we used whole genome sequencing raw reads and bioinformatics analysis to sequence and assemble Alectoris chukar's complete mitogenome for the first time. We also studied the maternal lineage and phylogenetic position of Alectoris chukar, as well as some mitogenomic diversity parameters. As a result, the complete mitogenomes with a length ranging from 16686 bp to 16688 bp of four individuals of wild Alectoris chukar were sequenced and assembled. They have a typical avian mitogenome structure with 2 ribosomal RNA (rRNA), 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and one non-coding control region. Our findings from bioinformatics analysis remarkably demonstrated that copies of the mitogenome are more abundant in liver tissues than in blood and in the liver tissues of females than in males. The results of phylogenetic analysis clustered the studied mitogenomes with Alectoris chukar as a monophyletic clade. Moreover, in comparison to the different genera, Alectoris chukar showed a high level of mitogenomic similarities to the snowcock species of the genus Tetraogallus within the Phasianidae family. However, they were more distant from other partridges. Additionally, a high percentage of mitogenomic pairwise identities within Iraqi Alectoris chukar and high mitogenomic variations compared to Chinese populations were discovered. The number and location of polymorphic sites indicated that the majority of the mitogenome sequences were conserved, with the control region, ND5, and CYTB genes having the most polymorphic sites. Analyses of phylogenetic and mitogenomic diversity revealed that samples of Alectoris chukar from Iraq have a unique maternal lineage and mitogenomic diversity specific to their geographic distribution, suggesting an Alectoris chukar kurdestanica subspecies. The molecular findings presented here provide valuable knowledge and mitogenomic resources into the evolutionary relationships of Alectoris chukar from the Middle East to avian species in the Phasianidae family.
 Key words: DNA sequencing; Mitochondrial genome; Partridges; Phasianidae; Phylogeny

Molecular epidemiological characteristics and genetic evolutionary relationships of methicillin-resistant Staphylococcus aureus of different avian origins in Qingdao, China, using whole-genome sequencing.

To understand the prevalence of avian methicillin-resistant Staphylococcus aureus (MRSA) and the current status of drug resistance in Qingdao, a comprehensive molecular epidemiological investigation and analysis of evolutionary relationships of MRSA isolates from broiler and layer chickens and waterfowl was conducted. One hundred and two avian MRSA strains were identified by multi-locus sequence typing, staphylococcal protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, and whole-genome sequencing. The sequence type (ST) 9-t899-SCCmec IVb type represented the highest proportion of avian-derived MRSA strains (71.57%), with ST398 type strains occasionally observed in broilers and waterfowl. The poultry-derived MRSA strains were all resistant to eight or more antimicrobials. Avian-derived MRSA strains carried 20 resistance genes, 109 virulence genes and 10 plasmids. Strains carrying the cfr oxazolidinone resistance gene were occasionally seen in broiler- and layer-derived MRSA. Single nucleotide polymorphism (SNP) core genome evolution and locus difference analysis showed that the closest strains were all of ST9-t899 type (to which also affiliated the highest number of strains) and this type occurred on all three kinds of poultry farm, but the SNP difference loci between strains of the same type ranged from 0 to 1472. The dominant type of MRSA from different poultry sources in Qingdao is ST9-t899-SCCmec IVb, which is commonly resistant to a variety of antimicrobial drugs and carries a variety of resistance genes and a large number of virulence genes. Sequence type 9-t899 type is widely spread among the three kinds of poultry investigated, but there are differences in affiliations.

42 Introgression within the horse's genome

Introgression is the transfer of genetic material between 2 different species via repeated back crossing of the interspecific hybrid and one of the parent species. These are important events to understand in the study of molecular evolutionary relationships between species. Previous studies have found regions of introgression within the horse genome, likely hundreds of thousands to millions of years ago from a non-caballine equid that are present in current horse populations. Our hypothesis is, these regions are retained among horses because they are doing something functionally beneficial and are increasing the horse's fitness. After identifying these regions with maximum likelihood estimation in a sample Thoroughbred population, the next steps were to analyze the different haplotypes found in modern horse genomes to find what polymorphisms were inherited together. Once identified, the genotypes of 230 Thoroughbred horse genomes were phased with BEAGLE. The positions tagged by BEAGLE were then viewed in Tassel 5 and a linkage disequilibrium plot was created. The phased genotypes were then analyzed, and the different haplotypes were extracted from the variant call file (vcf), taking into consideration the linkage disequilibrium plot and allele frequencies. Three main haplotypes occurred in this sample population: the reference, orTwilight, haplotype, the non-caballine haplotype, and another haplotype of unknown origin. This pipeline presents the number of haplotypes in the sampled population and which polymorphisms are inherited together, which gives us a better understanding of the origin on the introgressed haplotype(s). The short-term goal is to identify which non-caballine equid is the most prominent donor of the non-caballine haplotype. Ultimately, we will develop software to automate this analysis and identify putatively impactful introgressed regions using recently published data from the Equine-FAANG project. Currently, we are finding what the introgressed regions code for in the context of the horse genome, either non-coding RNA, a gene, or a non-coding region, to narrow down the search when putting together phylogenetic trees. Overall, the more we understand introgression, the better we will be able to piece together evolutionary relationships, how they are essential for rapid adaptation historically, and how they impact gene function in modern populations.

Genome-Wide Analysis of the Expansin Gene Family in Populus and Characterization of Expression Changes in Response to Phytohormone (Abscisic Acid) and Abiotic (Low-Temperature) Stresses.

Expansins are a group of cell wall enzyme proteins that help to loosen cell walls by breaking hydrogen bonds between cellulose microfibrils and hemicellulose. Expansins are essential plant proteins that are involved in several key processes, including seed germination, the growth of pollen tubes and root hairs, fruit ripening and abscission processes. Currently, there is a lack of knowledge concerning the role of expansins in woody plants. In this study, we analyzed expansin genes using Populus genome as the study target. Thirty-six members of the expansin gene family were identified in Populus that were divided into four subfamilies (EXPA, EXPB, EXLA and EXLB). We analyzed the molecular structure, chromosome localization, evolutionary relationships and tissue specificity of these genes and investigated expression changes in responses to phytohormone and abiotic stresses of the expansin genes of Populus tremula L. (PtEXs). Molecular structure analysis revealed that each PtEX protein had several conserved motifs and all of the PtEXs genes had multiple exons. Chromosome structure analysis showed that the expansin gene family is distributed on 14 chromosomes. The PtEXs gene family expansion patterns showed segmental duplication. Transcriptome data of Populus revealed that 36 PtEXs genes were differently expressed in different tissues. Cis-element analysis showed that the PtEXs were closely associated with plant development and responses to phytohormone and abiotic stress. Quantitative real-time PCR showed that abscisic acid (ABA) and low-temperature treatment affected the expression of some PtEXs genes, suggesting that these genes are involved in responses to phytohormone and abiotic stress. This study provides a further understanding of the expansin gene family in Populus and forms a basis for future functional research studies.

Population structure and genetic diversity of tobacco mild green mosaic virus variants in Western Anatolia of Turkey

Tobamoviruses have been pioneers in understanding the population genetic structure, host-vector interactions, and evolutionary processes of viruses. To better understand the population genetic structure and molecular evolutionary relationships of tobacco mild green mosaic virus (TMGMV), a significant species of tobamoviruses, extensive analyses were performed using bioinformatics tools in this study. 300 samples were collected from plants exhibiting viruses and virus-like symptoms from Turkey's largest tobacco cultivation areas during the plant vegetation period between 2019 and 2020. Samples were tested using conventional molecular techniques for tobamoviruses, including ToBRFV, TMV, TMGMV, ToMV, and ToMMV species. Single and double tobamovirus infections were determined in 258 of 300 samples. Single infections were 225 (75%), 10 (3.33%), and 1 (0.33%) for TMGMV, TMV, and ToMV, respectively. Double infections were 21 (7.0%) and 1 (0.33%) for TMGMV + TMV and TMV + ToMV, respectively. TMGMV-infected samples from each region were chosen, and their complete genomes were revealed. Detailed analyses were carried out from Turkey, with global variants available in GenBank. Molecular evolutionary analyses revealed three main lineages (Clades I, II, and III) at the p126, p183, MP, and complete genome levels and two main lineages at the CP gene (Clades I and II). Variants from Turkey were distributed in two different branches in other phylogenetic trees, except for the CP gene. High haplotype diversity and low nucleotide diversity were determined in each gene region, indicating consistent genetic stability. In addition, purifying selection pressures were determined in specific gene regions. With AMOVA (analysis of molecular variance), it was determined that the source of genetic variation came from within the main lineages, including various hosts and geographies. The differences in lineages were confirmed by independent test statistics. While neutrality tests revealed population expansions in the CP and MP genes, other p183 and p126 genes revealed bottlenecks or balancing selection. The fact that TMGMV was more common than TMV in Turkey strongly supported the phenomenon called “mutational melting” or “Müller latch,” which presumably causes TMV to disappear from its niche.

Identification and Characterization of ZF-HD Genes in Response to Abscisic Acid and Abiotic Stresses in Maize

The zinc finger homeodomain (ZF-HD) genes belong to the homeobox gene family, playing critical roles in flower development and stress response. Despite their importance, however, to date there has been no genome-wide identification and characterization of the ZF-HD genes that are probably involved in stress responses in maize. In this study, 24 ZF-HD genes were identified, and their chromosomal locations, protein properties, duplication patterns, structures, conserved motifs and expression patterns were investigated. The results revealed that the ZF-HD genes are unevenly distributed on nine chromosomes and that most of these genes lack introns. Six and two ZF-HD genes have undergone segmental and tandem duplication, respectively, during genome expansion. These 24 ZF-HD transcription factors were classified into six major groups on the basis of protein molecular evolutionary relationship. The expression profiles of these genes in different tissues were evaluated, resulting in producing two distinct clusters. ZF-HD genes are preferentially expressed in reproductive tissues. Furthermore, expression profiles of the 24 ZF-HD genes in response to different kinds of stresses revealed that ten genes were simultaneously up-regulated under ABA, salt and PEG treatments; meanwhile four genes were simultaneously down-regulated. These findings will pave the way for deciphering the function and mechanism of ZF-HD genes on how to implicate in abiotic stress.

First Report and Comparative Genomic Analysis of Mycoplasma capricolum subsp. capricolum HN-B in Hainan Island, China

Mycoplasma capricolum subsp. Capricolum (Mcc) is an important member of the Mycoplasma mycoides cluster (Mm cluster) and causes caprine contagious agalactia. Mcc can infect goats of all age groups, especially pregnant ewes and kids. It can cause the abortion in pregnant ewes and the death of goat kids, leading to enormous losses in the goat breeding industry. To date, the prevalence of epidemic Mcc strains on Hainan Island, China, remains unclear. This study aimed to isolate and identify Mcc strains endemic to Hainan Island, China. Genome sequencing and comparative genomic analysis were performed to reveal the molecular characteristics and evolutionary relationships of the isolated strain. Mcc HN-B was isolated and identified in Hainan Island, China. The Mcc HN-B genome consists of a 1,117,925 bp circular chromosome with a 23.79% G + C content. It contains 912 encoding genes, 3 gene islands, and 14 potential virulence genes. The core genome with the features of the Mm cluster and the specific genes of Mcc HN-B were identified by comparative genomic analysis. These results revealed the evolutionary relationship between Mcc HN-B and other members of the Mm cluster. Our findings provide a reference for further studies on the pathogenic mechanism and local vaccine development of Mcc.

Molecular Characterization of Outer Capsid Proteins VP5 and VP7 of Grass Carp Reovirus.

Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and mammalian orthoreoviruses was revealed. However, the functions of the aquareovirus genome-encoded proteins are poorly understood. We investigated the molecular characteristics of the outer capsid proteins, namely, VP5 and VP7, of grass carp reovirus (GCRV). The peptides VP5 and VP7 were determined using in-gel tryptic digestion and mass spectrometry. Recovered peptides represented 76% and 66% of the full-length VP5 and VP7 sequences, respectively. Significantly, two-lysine acetylation, as well as two-serine and two-threonine phosphorylation modifications, were first revealed in VP5. We found that the initial amino acid in VP5 was Pro43, suggesting that a lower amount of VP5 remained uncleaved in virions at the autocleavage site (Asn42-Pro43). Further biochemical evidence showed that the cleaved VP5N/VP5C conformation was the major constituent of the particles. Moreover, early cleavage fragments of VP7 and enhanced infectivity were detected after limited tryptic digestion of GCRV, indicating that stepwise VP7 cleavage is essential for VP5 conformational rearrangement. Our results provide insights into the roles of posttranslational modifications in VP5 and its association with VP7 in the viral life cycle.

Oxidosqualene cyclases in triterpenoids biosynthesis: a review

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

Molecular Evolution of Lysine Biosynthesis in Agaricomycetes.

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.

Comparative analysis of codon usage patterns of FUT2 from different species

Enterotoxigenic E. coli is an important zoonotic pathogen causing diarrhea in human and newborn animals. α - (1,2) fucosyltransferase 2 (FUT2) is closely associated with the formation of pathogenic receptors of Enterotoxigenic E. coli. Codon usage bias analysis can help to better understand the molecular mechanisms and evolutionary relationships of a particular gene. In order to understand the codon usage pattern of FUT2 gene, FUT2 gene coding sequences of nine species were selected from GenBank database for calculating the nucleotide composition (GC content) and genetic indices including effective number of codons, relative synonymous codon usage and relative codon usage bias using R software, in order to analyze codon usage bias and base composition in FUT2 gene from different species. The results showed that the codon usage of FUT2 gene in different species was affected by GC bias, especially GC frequency at the third position of codon (GC3). Most of the optimal codons were biased towards the G/C-ending types. GCC, CUG, UCC, GUG and AUC showed the highest relative synonymous codon usage value among different species, belonging to the most dominant codons. The usage characteristic of the codens for FUT2 gene in Sus scrofa was similar to that of Bos taurus; Homo sapiens was similar to Pan troglodytes. Effective number of codons was significantly, negatively correlated with GC3, and the relative higher frequency of optimal codon implied that FUT2 genes from different species had a strong bias in codon usage.

Investigations on annual spreading of viruses infecting cucurbit crops in Uttar Pradesh State, India

During 2018 an intensive study was conducted to determine the viruses associated with cucurbitaceous crops in nine agroclimatic zones of the state of Uttar Pradesh, India. Total of 563 samples collected and analysed across 14 different cucurbitaceous crops. The results showed the dominance of Begomovirus (93%) followed by Potyvirus (46%), cucumber green mottle mosaic virus (CGMMV-39%), Polerovirus (9%), cucumber mosaic virus (CMV-2%) and Orthotospovirus (2%). Nearly 65% of samples were co-infected with more than one virus. Additionally, host range expansion of CMV, CGMMV and polerovirus was also observed on cucurbit crops. A new potyvirus species, zucchini tigre mosaic virus, earlier not documented from India has also been identified on five crops during the study. Risk map generated using ArcGIS for virus disease incidence predicted the virus severity in unexplored areas. The distribution pattern of different cucurbit viruses throughout Uttar Pradesh will help identify the hot spots for viruses and will facilitate to devise efficient and eco-friendly integrated management strategies for the mitigation of viruses infecting cucurbit crops. Molecular diversity and evolutionary relationship of the virus isolates infecting cucurbits in Uttar Pradesh with previously reported strains were understood from the phylogenetic analysis. Diverse virus infections observed in the Eastern Plain zone, Central zone and North-Eastern Plain zone indicate an alarming situation for the cultivation of cucurbits in the foreseeable future.

Molecular Diagnosis and Evolutionary Relationship Analysis Of Plant Parasitic Tea Garden Nematodes From Different Tea Estates In Sylhet Region of Bangladesh

Nematodes from plant-parasitic sources are ever-present and incidental to plant growth as well as crop production. The damage of tea gardens caused by nematode is often non-specific and easily confused with symptoms. The present study determined the parasitic and non-parasitic nematodes population in different tea gardens of the Sylhet region by their morphological and partial molecular characterization. Out of 13 tea gardens, it was observed that BTRI, Karimpur, Mathiura, and Tarapur tea garden has the highest number of parasitic and non-parasitic nematodes. After PCR amplification, DNA bands with desired amplicon size were detected by gel electrophoresis. Among thirteen soil samples, nematodes from Malnichara, Karimpur, BTRI, Mathiura , and Finlay had partially confirmed the presence of rootknot nematode (Meloidogyne spp.), root-lesion nematode (Pratylenchus brachyurus), burrowing nematode (Radopholus similis), reniform nematode (Rotylenchulus reniformis) and lance nematode (Hoplolaimus columbus) consequently based on approximately base pair of 1.7, 1.1 and 0.52 kb (different Meloidogyne spp.) 0.52, 0.52, 0.25 and 2.3 kb of specific genes. From evolutionary analysis, it might be said that Meloidogyne species are strongly related with each other making clusters except Meloidogyne natalie where this one is closely related with Hoplolaimus columbus in their evolutionary relationship as remaining others (Rotylenchulus reniformis, Radopholus similis, Pratylenchus brachyurus) are in different clusters in the same clade and this result could be confirmed after sequencing. J. Bio-Sci. 29(1): 101-109, 2021 (June)

Antigenic and Molecular Characterization of Virulent Newcastle Disease Viruses Circulating in Ethiopia Between 1976 and 2008.

IntroductionNewcastle disease virus (NDV) cultures held in the isolate collections in Ethiopia between 1976 and 2008 were not characterized using biological and molecular techniques. The already characterized NDV isolates belonged to genotype VI but the genetic nature of previously collected isolates, which could shade light on the history of introduction into the country and their evolutionary relationships, were not established.MethodsA total of 14 NDVs (11 obtained from outbreak cases in chickens and three commercial vaccinal strains used in the country) were inoculated into specific pathogen free (SPF) embryonated chicken eggs (ECE). Allantoic fluids harvested from grown SPF ECE were tested by heamagglutination (HA) and heamagglutination inhibition (HI) tests. Partial F gene sequences were generated for all samples and molecular evolutionary relationships were reconstructed together with reference sequences freely available online. The pathogenicities of the isolates were assessed in vivo by determining their intracerebral pathogenicity index (ICPI) in day-old chicks and molecularly by determination of F gene cleavage sites.ResultsOf these, 12 viruses (two vaccines and 10 outbreaks) were successfully propagated as evidenced by a positive heamagglutination (HA) test. These 12 propagated viruses were further characterized by heamagglutination inhibition (HI) test, of which only three viruses reacted with monoclonal antibody (MAb 617/616) specific for pigeon paramyxovirus-1. In addition, all 14 viruses were characterized by partial fusion (F) gene sequencing and phylogenetic tree reconstruction. The Ethiopian NDV isolates clustered with genotype VI viruses, forming two clades (groups 1 and 2) that have ancestral relationships with Egypt-1990 and Sudan-1975 like viruses.DiscussionThe characterized genotype VI NDVs were genetically similar to currently circulating NDVs in Ethiopia. The isolates had cleavage sites consistent with mesogenic/velogenic NDV with a mean ICPI value of 1.76, indicating that the isolates were velogenic. Two and four highly virulent viruses were thermostable at 56°C for 2 hours and 1 hour, respectively. To reduce chicken mortality and production losses, proper control of the disease should be instituted using high quality and protective vaccines together with strong biosecurity measures.

Multi-epitope based peptide vaccine design using three structural proteins (S, e, and m) of sars-cov-2: An in silico approach

Introduction: The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has jeopardized our health system and leaving everyone in disarray. Despite the diligent cumulative effort of academia, there is hardly any light in the end tunnel so far in developing efficient and sustainable treatment options to tackle this public health threat. Therefore, designing a suitable vaccine to overcome this hurdle calls for immediate attention. The current study aimed to design a multi-epitope based vaccine using immunoinformatics tools. Materials and Methods: We approached the structural proteins: S, E, and M proteins of SARS-CoV-2 since they facilitate the infection of the virus into a host cell. By using different bioinformatics tools and servers, the multiple B-cell and T-cell epitopes were predicted potential for the required vaccine design. The phylogenetic analysis provides in-depth knowledge on ancestral molecular changes and the molecular evolutionary relationship of S, E, and M proteins. Results: Based on the antigenicity and surface accessibility of the spike (S), envelope (E), and membrane (M) proteins, eight epitopes were selected by various B cell and T cell epitope prediction tools. Molecular docking was executed to interpret the binding interactions of these epitopes from where three potential epitopes WTAGAAAYY, YVYSRVKNL, and GTITVEELK were finalized with their noticeable higher binding affinity scores-9.1,-7.4, and-7.0 kcal/mol, respectively. It is noteworthy to mention that the targeted epitopes are believed to cover 91.09% of the population coverage worldwide. Conclusions: In sum, we identified the three most potential epitopes at length, which might be turned to our purpose of designing the peptide-based vaccine against SARS-CoV-2. © 2021 The Author(s).

Molecular evolutionary relationships of Vietnamese and global pulmonary Paragonimus species in the family Paragonimidae and suborder Xiphidiata (Platyhelminthes: Trematoda)

Paragonimiasis, caused by Paragonimus species belonging to the family Paragonimidae of the suborder Xiphidiata (Platyhelminthes: Trematoda), often occurs in poor, upland, ethnic minorities, in Vietnam and the world. Asian Paragonimus species are distributed from Japan, South Korea, along with North and Southeast China, North-West and Central Vietnam, the Philippines, Thailand, Bangladesh, India, and Sri Lanka. There are various genetic variants, strains, and genotypes forming different complexes and evolutionary lineages. The 18S, 28S rDNA sequences and the intergenic transcribed spacer regions (ITS-1, ITS-2) of nuclear ribosomal transcription units are commonly used as molecular markers in genetic studies and phylogenetic analyses. We obtained a portion of 28S rDNA (domains D1–D3) of Paragonimus spp. including P. heterotremus (from Vietnam), P. ohirai (Japan), P. iloktsuenensis (Japan), and P. westermani (India and Vietnam) and conducted phylogenetic analysis for molecular evolutionary studies. The results showed that the family Paragonimidae formed the biggest cluster in a phylogenetic tree, which comprises of 46 sequences of 11 species belonging to 11 subgroups, among which the P. westermani complex of strains originating from China, Korea, Japan, India, Philippines, Malaysia, and Vietnam is present. P. westermani complex is arranged in a position of "sister" (sister group) with the subgroup P. siamensis. The P. heterotremus and P. ohirai complexes, and the P. miyazakii, P. harinasutai, P. mexicanus, P. kellicotti, and P. macrorchis complexes are clustered in a common population. P. westermani of Vietnam is in close proximity to the East Asian strains, as of which has been previously reported. P. ohirai and P. iloktsuenensis are considered “sibling” species, sharing the same clade. Phylogenetic analysis using the 28S rDNA sequences directly presented species position and their molecular evolutionary relationships in the families Paragonimidae, Troglotrematidae, Nanophyetidae, and Collyriclidae. Evolutionary analysis has also clarified a number of complex delineation problems and made a clear nomenclature for Paragonimus sp. of Vietnam, in particular, which has scientific grounds merited to recognize as that it is really the P. westermani species.

Bipartite molecular approach for species delimitation and resolving cryptic speciation of Exobasidium vexans within the Exobasidium genus

Exobasidium vexans, a basidiomycete pathogen, is the causal organism of blister blight disease in tea. The molecular identification of the pathogen remains a challenge due to the limited availability of genomic data in sequence repositories and cryptic speciation within its genus Exobasidium. In this study, the nuclear internal transcribed spacer rDNA region (ITS) based DNA barcode was developed for E. vexans, to address the problem of molecular identification within the background of cryptic speciation. The isolation of E. vexans strain was confirmed through morphological studies followed by molecular identification utilizing the developed ITS barcode. Phylogenetic analysis based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) confirmed the molecular identification of the pathogen as E. vexans strain. Further, BI analysis using BEAST mediated the estimation of the divergence time and evolutionary relationship of E. vexans within genus Exobasidium. The speciation process followed the Yule diversification model wherein the genus Exobasidium is approximated to have diverged in the Paleozoic era. The study thus sheds light on the molecular barcode-based species delimitation and evolutionary relationship of E. vexans within its genus Exobasidium.