Molecular Evolutionary Genetics Analysis Research Articles

Molecular Identification of Dominant Microbes in Kola nut (Cola nitida)

Kola nut is an important dehiscent fruit but its supply fails to meet the demand due to limitations such as inadequate post-harvest practices leading to spoilage during transportation and storage. Identification of microbial contaminants is a necessary step in reducing the loss. Hence, this work aimed at characterizing the genome of dominant fungus and bacteria associated with kola nut (Cola nitida). Samples of kola nuts were collected from Oje and Oja-oba markets in Ibadan, Nigeria. Isolates were extracted from the lesion region of the samples using the standard method. Macro-morphological and micro-morphological observations of the pure culture of the isolates were conducted. After genomic DNA extraction of fungal and bacterial isolates, Polymerase Chain Reaction (PCR) was carried out using Internal Transcribed Sequence (ITS 1 and ITS 4) and 27F primers for fungal and bacterial genome respectively. Sanger sequencing of the purified DNA fragments was conducted utilizing the Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000. Molecular Evolutionary Genetics Analysis (MEGA version 6) was used for phylogenetic analysis. The result revealed that one fungus and two bacteria were associated with kola nuts. The fungal isolate showed 100% identity with Aspergillus wentii from the National Center for Biotechnology Information data base. The fungus showed a good relationship with A. dimorphicus and A. europaeus. The two bacterial isolates were also identified through the phylogenic analysis as Bacillus cereus and B. subtilis. B. cereus is closely related to B. thuringiensis. This research provides background information on the Kola nut microbial contaminant. Keywords: Bacteria, Contaminant, Fungus, Identification, Primers, Sequencing

Analyses and Insights into Genetic Reassortment and Natural Selection as Key Drivers of Piscine orthoreovirus Evolution.

Piscine orthoreovirus (PRV) is a pathogen that causes heart and skeletal muscle inflammation in Salmo salar and has also been linked to circulatory disorders in other farmed salmonids, such as Oncorhynchus kisutch and Oncorhynchus mykiss. The virus has a segmented, double-stranded RNA genome, which makes it possible to undergo genetic reassortment and increase its genomic diversity through point mutations. In this study, genetic reassortment in PRV was assessed using the full genome sequences available in public databases. This study used full genome sequences that were concatenated and genome-wide reassortment events, and phylogenetic analyses were performed using the recombination/reassortment detection program version 5 (RDP5 V 5.5) software. Additionally, each segment was aligned codon by codon, and overall mean distance and selection was tested using the Molecular Evolutionary Genetics Analysis X software, version 10.2 (MEGA X version 10.2). The results showed that there were 17 significant reassortment events in 12 reassortant sequences, involving genome exchange between low and highly virulent genotypes. PRV sequences from different salmonid host species did not appear to limit the reassortment. This study found that PRV frequently undergoes reassortment events to increase the diversity of its segmented genome, leading to antigenic variation and increased virulence. This study also noted that to date, no reassortment events have been described between PRV-1 and PRV-3 genotypes. However, the number of complete genomic sequences within each genotype is uneven. This is important because PRV-3 induces cross-protection against PRV-1, making it a potential vaccine candidate.

Exploring the Relationship Between Growth Hormone Secretagogue Receptor (GHSR) Gene and Body Proportions in Bangkok Chickens: Insights from DNA Sequencing and Hin6I Enzyme-Restricted PCR-RFLP Analysis

The purpose of this study was to determine the diversity of the growth hormone secretagogue receptor (GHSR) gene and also to analyze the association of the GHSR gene with the body proportions of Bangkok chickens. A total of 125 Bangkok chickens were reared, and blood samples were taken. Bangkok chickens were kept in colony cages with ad libitum feeding and drinking. The GHSR gene polymorphism was determined using the PCR-RFLP method. The restriction enzyme used in this study was Hin6I. The data analyzed were indicators of body proportions, which included data on body weight, carcass weight, and commercial cut weight. Single nucleotide polymorphism (SNP) identification using the Molecular Evolutionary Genetics Analysis 7 program with reference number AB095994.1. Allele frequency values, genotype frequencies, and Hardy-Weinberg balance values were also analyzed. The association between the GHSR gene and the traits observed in Bangkok chickens was analyzed using the T-test. The results of the study showed that the GHSR gene in Bangkok chickens had two genotypes, namely TT and CT. The values for Ho and He were 0.224 and 0.198, respectively. The genotype frequencies of TT and CT were 0.776 and 0.224, respectively. The two genotypes were associated with body weight, carcass weight, and commercial weight (p < 0.05). For all measured criteria, the TT genotype showed a higher weight value than the CT genotype. The GHSR gene has the potential to be used as a genetic marker for the selection process on body weight, carcass weight, and commercial weight traits.

Disappearance of Imported Cases of Omicron Lineage BA.2.40 in West Kalimantan, Indonesia.

The World Health Organization has declared Omicron as the fifth variant of concern with more than 50 mutations, particularly in the spike protein. Given increased viral infectivity due to mutations, worldwide genomic surveillance and detection of severe acute respiratory syndrome 2 (SARS-CoV-2) is essential. The present study aimed to track Omicron lineage BA.2.40 in West Kalimantan, Indonesia. In May-August 2022, nasopharyngeal swab samples (n=3,642) were collected from international travelers to West Kalimantan (active surveillance), and patients hospitalized due to SARS-CoV-2 infection (baseline surveillance). The samples were tested for Omicron lineages based on ORF1ab, N, and HV69-70del genes, followed by whole-genome sequencing. The sequences were then identified using two genomic databases, aligned against the reference genome (Wuhan/Hu-1/2019), and then compared with BA.2.40 lineage detected across the world. Phylogenetic analysis between the samples and other SARS-CoV-2 isolates was performed using molecular evolutionary genetics analysis software. Based on the genomic databases, 10 isolates were identified as BA.2.40. All samples tested positive for the ORF1ab and N genes, but negative for the HV69-70del gene, which is a marker to detect the Omicron variant. Phylogenetic analysis showed the isolates were closely related to an isolate from Malaysia, an area dominated by BA.2.40. Omicron lineage BA.2.40 has no HV69-70 deletion in the spike protein, a marker used to screen for the Omicron variant. BA.2.40 showed a high similarity to an isolate from Malaysia and was detected only during certain periods, indicating the effect of internationally imported cases.

Diversity of astroviruses in wild animals in Yunnan province, China

BackgroundAstroviruses (AstVs) are single-stranded RNA viruses that have been detected in a wide range of mammals and birds. They are associated with numerous interspecies transmissions and viral recombination events, posing a threat to human and animal health.MethodsWe collected 1,333 samples from wild animals, including bats, rodents, wild boars, and birds, from various states and cities in the Yunnan Province, China, between 2020 and 2023 to investigate the presence of AstVs. AstVs were detected using a polymerase chain reaction targeting the RdRp gene. Finally, the Molecular Evolutionary Genetics Analysis software was used to construct the phylogenetic tree.ResultsThe overall positivity rate for AstVs was 7.12% in four species, indicating their widespread occurrence in the region. High genetic diversity among AstVs was observed in different animal species, suggesting the potential for interspecies transmission, particularly among rodents and birds. Additionally, we identified a novel AstV strain and, for the first time, provided information on the presence of bastroviruses in Yunnan, China.ConclusionsThe widespread distribution and high genetic diversity of AstVs, along with the observed potential for interspecies transmission, highlight the importance of further investigation and surveillance in the region. The findings emphasize the need for increased attention to AstVs and their potential impact on human and animal health in Yunnan and other regions.

Avian infectious bronchitis virus (AIBV) review by continent.

Infectious bronchitis virus (IBV) is a positive-sense, single-stranded, enveloped RNA virus responsible for substantial economic losses to the poultry industry worldwide by causing a highly contagious respiratory disease. The virus can spread quickly through contact, contaminated equipment, aerosols, and personal-to-person contact. We highlight the prevalence and geographic distribution of all nine genotypes, as well as the relevant symptoms and economic impact, by extensively analyzing the current literature. Moreover, phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA-6), which provided insights into the global molecular diversity and evolution of IBV strains. This review highlights that IBV genotype I (GI) is prevalent worldwide because sporadic cases have been found on many continents. Conversely, GII was identified as a European strain that subsequently dispersed throughout Europe and South America. GIII and GV are predominant in Australia, with very few reports from Asia. GIV, GVIII, and GIX originate from North America. GIV was found to circulate in Asia, and GVII was identified in Europe and China. Geographically, the GVI-1 lineage is thought to be restricted to Asia. This review highlights that IBV still often arises in commercial chicken flocks despite immunization and biosecurity measures because of the ongoing introduction of novel IBV variants and inadequate cross-protection provided by the presently available vaccines. Consequently, IB consistently jeopardizes the ability of the poultry industry to grow and prosper. Identifying these domains will aid in discerning the pathogenicity and prevalence of IBV genotypes, potentially enhancing disease prevention and management tactics.

Mycotoxic effects of entomopathogenic fungi of fall armyworm (Spodoptera frugiperda J.E. Smith) on poultry feed safety

This study was carried out to analyze mycotoxins of entomopathogenic fungi of fall armyworm (Spodoptera frugiperda J.E. Smith) and poultry feed safety. An experiment was set up to assess the types of mycotoxins produced by entomopathogenic fungal parasite of fall armyworm larvae and their subsequent effect on the safety of the larvae as a feed ingredient. Molecular characterization was done to estimate the diversity of entomopathogenic fungi on fall armyworm larvae specimens from the treatment plots. Sequenced data was analyzed and processed using Molecular Evolutionary Genetic Analysis 6.0 software. The results showed relative diversity of fall armyworm larvae with 11 species isolated belonging to Aspergillus, Penicillium, Fusarium, Trichoderma, Bipolaris and Irpex genus. Some of these are potential mycotoxin producing fungi. Although isolated fungi potentially produce Ochratoxin, Fumonisin, Zearalenone and Trichothecene mycotoxins, only aflatoxins were analyzed in this study. About 3.98 μg /kg of aflatoxin was observed using the ELISA total assay which is within the threshold toxicity levels set in Kenya for feed of about 20 μg /kg. While this is under the threshold set by Kenya, it is still enough to cause concern as the cumulative exposure of even low doses can have impacts. This study therefore concludes that, Spodoptera frugiperda can be potentially contaminated with aflatoxins and when formulating poultry diets, there is need to monitor production so that the quality is not compromised and feed safety is ensured. Further studies are recommended to determine how much produced aflatoxins are then transferred into the poultry products such as eggs and meat.

PHYLOGENETIC RELATIONSHIP BETWEEN RABIES VIRUS (RABIES LYSSAVIRUS) VARIANTS FROM TWO DIFFERENT HOST SPECIES

Rabies is a fatal zoonotic disease that affects several mammals. Bats are recognized hosts of the rabies virus, and their main food source is the blood of other mammals, particularly cattle. During feeding, bats transmit the virus to cattle, which are victims of the disease, contributing to economic losses and increasing the risk of infection for humans. Based on this affinity in the rabies cycle between bats and cattle, the objective of this study was to analyze the phylogenetic relationships of rabies virus samples in cattle and bats. The G gene of the rabies virus was chosen for this study because it is directly related to the infection process. Nucleotide sequences of the viral G gene were selected from GenBank for samples obtained from infected cattle and bats. Maximum parsimony analyses were conducted using the Molecular Evolutionary Genetics Analysis software. The Maxima Parsimony tree indicated a phylogenetic relationship between the G genes of both hosts, indicating that the virus evolved from bats to cattle. Analysis of parsimoniously informative sites revealed that the viral G gene carried specific mutations in each host. Knowledge of the evolutionary relationships between the rabies virus and its hosts is critical for identifying potential new hosts and the possible routes of infection for humans.

Single Nucleotide Polymorphism R25C and R25H on Leptin gene and their association with body weight and measurements of Pasundan cattle

Abstract. Hilmia N, Ramdani D, Hidayat R, Widyastuti R, Hernaman I, Dudi, Edianingsih P, Arifin J. 2023. Single Nucleotide Polymorphism R25C and R25H on Leptin gene and their association with body weight and measurements of Pasundan cattle. Biodiversitas 24: 6310-6315. The Leptin gene is a potential candidate for genetic selection in livestock. Nonsynonimous Single Nucleotide Polymorphisms (SNP) Arg25Cys/R25C in Leptin gene associated with fat deposition mainly. This study aimed to identify polymorphism of Leptin Gene based on SNPs of Pasundan cattle and their association with body weight and measurements. This research used 64 DNA samples along with the corresponding body weight and measurement data from Pasundan cattle. About 9 and 7 DNA samples from Bali and Ongole cross (PO) cattle respectively, were also collected as comparisons. The DNA along 620 bp was multiplied by PCR. The SNPs in the Leptin gene on exon two were identified, followed by direct sequencing of the PCR product. The sequencing results were analyzed by BioEdit and Molecular Evolutionary Genetic Analysis (MEGA) 4.0. The General Linear Model analyzed the association between SNPs and body weight and measurements. The results showed there were two nonsynonymous SNP in the Leptin gene on exon two, i.e. g.1047C>T/Arg25Cys/R25C (T allele) that changed amino acid Arginine to Cysteine and g.1048G>A/Arg25His/R25H (A allele) which changed Arginine to Histidine. The frequency of the C allele in the Leptin gene on Pasundan cattle (0.54) was higher than mutation alleles, i.e. T ( 0.29) and A allele (0.17). There were six genotypes: CC (0.266), CT (0.390), CA (0.156), TT, TA, and AA 0.063 respectively. The Leptin gene mutation at the 25th position of amino acids, which changed Arginin to Cystein or Histidin (R25C and R25H), did not affect body weight and measurement in Pasundan Cattle. Therefore, nonsynonymous SNP R25C and R25H could not be used as marker genetic selection in Pasundan cattle.

Barkodlama Bölgesi Olarak Potentilla L. Cinsinde psbA-trnH IGS Sekanslarının Değerlendirmesi

Barkodlama Bölgesi Olarak Potentilla L. Cinsinde psbA-trnH IGS Sekanslarının Değerlendirmesi

Molecular cloning and heterologous expression analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase gene in Centella asiatica L.

Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.

Morphogenetic Sharks of Genus Carcharinus Traded at Southwest Aceh Fish Landing Sites

Research on the types of sharks traded at the Southwest Aceh fish landing site was conducted in September 2022. The purpose of this study is to analyze the morphology and genetic analysis of sharks. Sampling uses purposive sampling techniques, for morphological analysis refers to shark identification books, while genetic analysis uses the Sanger method used at the sequencing stage. Morphological data analysis using Image-j software, for genetic analysis, using Mega 6 (Molecular Evolutionary Genetic Analysis). Morphological analysis was carried out at the Laboratory of Biosystematics and Marine Genetics, Faculty of Fisheries and Marine Sciences, Teuku Umar University. Genetic analysis was carried out at the Denpasar Biodiversity Laboratory, Bali. The results of this study found two species of sharks with a total of 8 individuals from the genus Carcharinus

Protein sequence comparison based on representation on a finite dimensional unit hypercube

Numerous techniques are used to compare protein sequences based on the values of the physiochemical properties of amino acids. In this work, a single physical/chemical property value based non-binary representation of protein sequences is obtained on a 20 × 20-dimensional unit hypercube. The represented vector expressed in the matrix form is taken as the descriptor. The generalized NTV metric, which is an extension of the NTV metric used for polynucleotide space is taken as a distance measure. Based on this distance measure, a distance matrix is obtained for protein sequence comparison. Using this distance matrix, phylogenetic trees are drawn by using Molecular Evolutionary Genetics Analysis 11 (MEGA11) software applying the neighbor-joining method. Data sets used in this current work are 9-ND4, 9-ND5, 9-ND6, 24 TF-LF proteins, 27 different viruses and 127 proteins from the protein kinase C (PKC) family. Two sets of phylogenetic trees are obtained – one based on property value of polarity and the other based on property value of molecular weight. They are found to be exactly the same. Similar results also hold for other single property value based representation. The present trees are individually tested for efficiency based on the criterion of rationalized perception and computational time. The results of the present method are compared with those obtained earlier by other methods on the same protein sequences using assessment criteria of Symmetric distance (SD), Correlation coefficient, and Rationalized perception. In all the cases, the present results are found to be better than the results of other methods under comparison. Communicated by Ramaswamy H. Sarma

Molecular and Serological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Horses from the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease in East Asia caused by the SFTS virus (SFTSV). It is to investigate the presence of SFTSV RNA and antibodies in horses from a slaughterhouse and equestrian centers in the Republic of Korea (ROK). A prevalence study of SFTSV-specific RNA and antibodies was designed from 889 horses in the ROK. Materials and Methods: Serum samples were collected from horses at a slaughterhouse and equestrian centers from 2018 to 2020. To detect the presence of SFTSV, RNA was extracted from the serum samples, and a nested reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Sequencing data were analyzed, and a phylogenetic tree was constructed using the maximum-likelihood method with Molecular Evolutionary Genetics Analysis Version 7.0 software. The horse sera were also tested for SFTSV-specific immunoglobulin G antibodies using enzyme-linked immunosorbent assay (ELISA). Results: Twelve of 889 (1.3%) horse sera were positive for SFTSV RNA, and 452 of 887 (51.0%) horse sera were seropositive by ELISA. Among the RT-PCR-positive samples, 12 of the SFTSV S-segment sequences were classified as sub-genotypes B-2 (n = 6) and B-3 (n = 6). ELISA analysis was evaluated by comparison with neutralization test. We investigated SFTSV infection in horses over a 3-year period, but sampling was not performed evenly by season; continuous surveillance of SFTSV in horses is needed. Conclusions: We report the detection of SFTSV RNA and provide serological data on SFTSV prevalence in horses in the ROK. The detection of SFTSV-specific RNA and antibodies in horses, which are in close proximity to humans, suggests that SFTS is an emerging and important health issue, indicating that more attention to its relevance for equestrian workers is needed.

The sequence variation of mitochondrial tRNA tyrosine and cysteine among Iranian women with idiopathic recurrent miscarriage: A case-control study.

Recurrent miscarriage is one of the most prevalent reproductive diseases. This phenomenon has several reasons, including maternal, hormonal, immunological, and parental genetic factors. Idiopathic recurrent miscarriage (IRM), with no distinctive etiology, involves about half of the recurrent miscarriage cases. Some mutations in mitochondrial DNA can lead to miscarriage. Mitochondrial tRNA (mt-tRNA) mutations cause nearly half of the mitochondrial disorders. To identify mt- tRNA gene mutations in Iranian women with IRM. In this case-control study, 100 Iranian women with IRM and 100 women as control without any history of miscarriage were investigated by polymerase chain reaction-single strand conformation polymorphism technique followed by gene sequencing. Bioinformatics analysis were done using human mitochondrial genome database, molecular evolutionary genetics analysis, mammalian mitochondrial-tRNA, etc. Results showed 4 mt-tRNA mutations including 1 cysteine mt-tRNA mutation (5824CT) and 3 tyrosine mt-tRNA mutations (5868TA, 5849CT, and 5836TC) in our cases. Amongst the 4 mutations found, one was novel that is still not reported. Our bioinformatics analysis revealed that these mutations can be pathogenic. They occurred in tRNA-conserved regions and their secondary structure was changed, which can result in mitochondrial dysfunction. Mutations of these genes may help in the assessment of IRM. Further study of all 22 mt-tRNAs possible mutations is recommended to describe their etiologic role in IRM.

DGAT1 gene polymorphism and their association with fat deposition and carcass quality in Pasundan cattle of Indonesia

Abstract. Dudi D, Hilmia N, Khaerunnisa I, Mushawwir A. 2023. DGAT1 gene polymorphism and their association with fat deposition and carcass quality in Pasundan cattle of Indonesia. Biodiversitas 24: 4202-4208. The bovineAcyl-CoA: Diacylglycerol O-acyltransferase 1 (DGAT1) gene is crucial to milk and meat quality in cattle. The K232A DGAT1 mutation was broadly used as a milk and meat quality genetic marker. Pasundan cattle are Indonesian local cattle from West Java. These local cattle have adapted to the tropical environment, environmental factors are not obstructing their development. The DGAT1 gene polymorphism information and their contribution to fat deposition and carcass quality in Pasundan cattle is very limited. The objective of this study was to examine the genetic polymorphism of the K232A DGAT1 gene and its association with fat deposition and carcass quality in Pasundan cattle population. The gene polymorphisms were identified using PCR, and direct sequencing to discover a single nucleotide polymorphism (SNP). All sequencing results (ABI trace files) were analyzed in FinchTV, BioEdit, and Molecular Evolutionary Genetic Analysis (MEGA) 6.0. Genotyping was performed on 80 Pasundan cattle. In comparison, Ongole-Grade (n=5), Bali (n=2), Simmental (n=2), Limousin (n=2), Madura (n=2), and Pesisir (n=2) were also used in this study. Their association with fat deposition and carcass quality was evaluated on seven heads of AK genotype and 23 heads of KK genotype. According to the sequencing result, two SNPs were found at g.201G>A and g.202C>A, respectively. The K232A DGAT1 locus was polymorphic in Pasundan cattle population with the K and A allele frequencies of 0.956 and 0.044, respectively. In addition, DGAT1 gene polymorphisms are not associated with back fat thickness, longissimus dorsi area, rump area, and intramuscular fat.

First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 μm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 μm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

Molecular characterization of rabies virus in trade dogs from Plateau state, Nigeria

Rabies is one of the most dreaded diseases known to mankind and it is endemic in Nigeria. This study was aimed at the detection and molecular characterisation of the rabies virus antigen in trade dogs in Plateau State. A cross-sectional study was performed to determine the prevalence of rabies antigen in the brain tissues of slaughtered dogs, from February to June 2018 using a direct fluorescent antibody test and mouse inoculation technique. Direct Fluorescent Antibody Test, Polymerase Chain Reaction (PCR), sequencing and phylogenetic analysis of the identified rabies virus were also undertaken. Phylogenetic analysis was done using molecular evolutionary genetic analysis (MEGA) 10 to determine their evolutionary relationship with other rabies virus sequences deposited in Genbank. Results indicated, out of 450 dog brain samples tested 14(3.1%) were positive for rabies virus antigen. Zoographic data obtained showed 66.3% of dogs were female, and the dogs were kept mainly for security (n=54) and breeding (n=28). Fourteen positive samples were used to inoculate 70 adult mice, only 8 samples were positive and 2 samples were also positive by RT PCR. Phylogenetic analysis of the nucleotide sequences showed that the two rabies virus sequences in this study, sequence MZ148311 had very close identity with EU038106 (99.8% homology) and sequence MZ148312 had perfect identity with EU038087 (100% homology) was most closely related to lyssavirus that has been reported to be circulating previously in Plateau and Nigeria, belonging to the Africa 2 lineage.

Molecular characterization and epidemiology of methicillin-resistant Staphylococcus aureus isolated from clinical samples in Sokoto, Nigeria

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health threat and a cause of hospital-acquired and community-acquired infections. This study was undertaken to investigate antimicrobial resistance pattern, the genetic lineage, presence of S. aureus protein A (SPA) gene, and staphylococcal chromosomal cassette mec (SCC mec) types of MRSA isolated from clinical samples sent for microbiological test in major government hospitals in Sokoto. Material and Methods: A total of 90 S. aureus MRSA isolates were collected and confirmed using standard microbiological techniques. Antibiotic susceptibility testing was done using the disk diffusion method; mecA detection and sequencing were carried out. Phylogenetic reconstruction was also done using the molecular evolutionary genetics analysis X software and phylogeny tree constructed by Neighbor-Joining method. SCC mec typing and SPA detection were also done. Results: Of the 90 S. aureus isolates, 42 were found to be MRSA using the cefoxitin disk diffusion, the most potent antibiotic against them was quinupristin/dalfopristin with 83.3% followed by rifampicin with 81.0% and 6 clindamycin with a 71.4%. With 78.6% of the isolates showing resistance to the fluoroquinolone antibiotic ciprofloxacin, tetracycline and gentamicin came in second and third, with 64.3% and 61.9% of isolates showing resistance, respectively. Most of the MRSA isolates were resistant to more than three antibiotics. Polymerase chain reaction showed 36 (85.7%) harbored the mecA gene and of the 36 mecA positive isolates, phylogenetic reconstruction of representative MRSA sequences showed that MRSA sequences in this study clustered in two closest clades suggesting a possible horizontal transfer. Of the 36 isolates, 23 were SCC mec type I, ten were type IV, and three were non-typeable, while the SPA gene was detected in all the isolates amplified. Conclusion: The use of phenotypic and molecular methods in this study provided useful information on antibiotic resistance profile, epidemiology, and molecular characteristics of MRSA isolates in Sokoto Nigeria. The information provided could help in monitoring the evolution of MRSA strains in Nigeria over time.

First record of the complete mitochondrial genome of Culex pipiens (Diptera: Culicidae) in Iraq and its phylogenetic analysis

The common house mosquito, Culex pipiens, is a worldwide insect that is known as a vector of several human and animals’ pathogens that cause critical diseases, such as malaria, yellow fever and encephalitis. The mitochondrial genome knowledge can supply a foresight for evolutionary biology and phylogenetic analysis. Thus, in the current study, the complete mitochondrial genomes of the Iraqi C. pipiens were first sequenced, assembled, annotated, and analyzed using high throughput sequencing techniques. The results revealed that the length of the complete mitochondrial genome is 14,856 bp, comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The A + T content was 77.09%, while the G+C content was only 22.1%. All PCGs open with the start codon ATN, except the cox1 gene and end with the stop codon TAA. A phylogenetic relatedness with other different species of Culex spp. was achieved by operating the molecular evolutionary genetic analysis based on the whole mitochondrial genome sequences. The results showed that C. pipiens from Iraq shares a close ancestry with C. pipiens from Tunisia and France. To our knowledge, this is the first report of the complete mitochondrial genome sequence of the Iraqi C. pipiens. As a consequence, this conclusion may provide a new profound insight into the evolution of C. pipiens in Iraq.