Nucleotide Differences Research Articles

Evaluation of various distance computation methods for construction of haplotype-based phylogenies from large MLST datasets

Multi-locus sequence typing (MLST) is widely used to investigate genetic relationships among eukaryotic taxa, including parasitic pathogens. MLST analysis workflows typically involve construction of alignment-based phylogenetic trees – i.e., where tree structures are computed from nucleotide differences observed in a multiple sequence alignment (MSA). Notably, alignment-based phylogenetic methods require that all isolates/taxa are represented by a single sequence. When multiple loci are sequenced these sequences may be concatenated to produce one tree that includes information from all loci. Alignment-based phylogenetic techniques are robust and widely used yet possess some shortcomings, including how heterozygous sites are handled, intolerance for missing data (i.e., partial genotypes), and differences in the way insertions-deletions (indels) are scored/treated during tree construction. In certain contexts, ‘haplotype-based’ methods may represent a viable alternative to alignment-based techniques, as they do not possess the aforementioned limitations. This is namely because haplotype-based methods assess genetic similarity based on numbers of shared (i.e., intersecting) haplotypes as opposed to similarities in nucleotide composition observed in an MSA. For haplotype-based comparisons, choosing an appropriate distance statistic is fundamental, and several statistics are available to choose from. However, a comprehensive assessment of various available statistics for their ability to produce a robust haplotype-based phylogenetic reconstruction has not yet been performed. We evaluated seven distance statistics by applying them to extant MLST datasets from the gastrointestinal parasite Cyclospora cayetanensis and two species of pathogenic nematode of the genus Strongyloides. We compare the genetic relationships identified using each statistic to epidemiologic, geographic, and host metadata. We show that Barratt’s heuristic definition of genetic distance was the most robust among the statistics evaluated. Consequently, it is proposed that Barratt’s heuristic represents a useful approach for use in the context of challenging MLST datasets possessing features (i.e., high heterozygosity, partial genotypes, and indel or repeat-based polymorphisms) that confound or preclude the use of alignment-based methods.

Morphology and molecular phylogeny of two new Aspidisca species (Ciliophora, Spirotrichea, Euplotida) collected from subtropical coastal waters in China

Ciliates are morphologically diverse and highly specialized unicellular eukaryotes that constitute an important component of the microbial food web. Aspidisca Ehrenberg, 1830, is a highly speciose genus that plays key ecological roles in a wide range of environments, and its species diversity has always been a hot spot in protozoan research. In this study, we investigate the living morphology, infraciliature, silverline system, and ribosomal small subunit (SSU rRNA) gene sequences of two new Aspidisca species collected from subtropical coastal waters of China using standard methods. Aspidisca spina sp. n. is characterized by having an obvious peristomial spur, two transparent posterior protrusions, seven frontoventral cirri in “polystyla-arrangement”, and six dorsal ridges. It can be distinguished from the most similar congener, A. magna Kahl, 1932, by the presence of posterior protrusions, more membranelles in the posterior part of adoral zone (AZM2), and different SSU rRNA gene sequences. A. shini sp. n. is smaller in body size, only 35–40 × 25–30 µm in vivo, with four prominent ridges on the arched dorsal side, very similar with the “well-known” species A. steini Buddenbrock, 1920, in morphological characteristics, but it can be distinguished by the arrangement of frontoventral cirri and 121 nucleotide difference in the SSU rRNA gene sequences. Phylogenetic analyses based on the SSU rRNA gene sequences revealed the systematic positions of two new taxa and supported the validity of them as distinct species.

Molecular genetic analysis reveals a novel B variant allele at the ABO locus

BackgroundWhen discrepancies in ABO blood typing emerge, ABO subgroups should be considered. Serological procedures such as the adsorption-elution test, the salivary ABH inhibition test, and the antiA1 (lectin) saline technique might be employed. These serological approaches, however in rare blood types are difficult to understand. As a result, a dependable and cost-effective approach for determining ABO subgroups is of special relevance. Methodologyfor this purpose, DNA sequencing is most reliable solution for accurate results. Exon 1 to 7 5′ 3’ UTR and intron 6 of ABO gene of a Chinese individual was sequenced because the laboratory was unable to determine the blood group by traditional method. ResultsThe analysis of the blood showed weak B antigens on their surface. Gene sequencing results revealed a novel B allele, that has one novel identified nucleotide difference from T to C at nucleotide 425. ConclusionThe results reported novel B allele which cannot be confirmed by conventional serological methods. Owing to the peculiarity of ABO blood types of inheritance, findings on the incidence and molecular causes of rare blood groups in the Chinese population have substantial therapeutic implications and are worthy of additional investigation in the future.

Isolation, identification, and pathogenicity of a ALV-K strain from Chinese indigenous chicken breed

Subgroup K avian leukosis virus (ALV-K) is a new subgroup of avian leukosis virus (ALV) first identified in Chinese indigenous chickens in recent years. In this study, an ALV-K strain was isolated from Luhua chicken in Shandong province, China, and designated SD20LH01. The full-length genomic sequence of SD20LH01 was 7491 bp, which had the highest homology with ALV-K reference strains GDFX0601, GDFX0602 and GDFX0603. The nucleotide homology of env gene of SD20LH01 with reference strains of subgroup A, B, C, D, E, and J was ranged from 57.1 to 93.2%, while 94.1 to 99.4% with other ALV-K reference strains. The nucleotide difference of SD20LH01 mainly clustered with gp85 gene and U3 sequence when compared with the reference strain of ALV-K. In order to investigate the pathogenicity of SD20LH01, SPF chicken embryos were infected by yolk sac inoculation, and 1-day-old chickens were infected by intraperitoneal inoculation of SD20LH01. The results showed that yolk sac inoculation of SD20LH01 could induce persistent viremia, growth retardation and reduce the immune response to NDV and AIV-H9 vaccines. However, intraperitoneal inoculation in 1-day-old chickens could only induce a low level of viremia. In addition, no tumors were found in infected chickens during the animal experiments. This study enriched the genomic sequence data of ALV-K isolated in Chinese indigenous chickens, and laid a foundation for further study on the pathogenesis and prevention of ALV-K.

Development of diagnostic SNP markers and a novel SNP genotyping assay for distinguishing opium poppies

The opium poppy acts as an important natural pain reliever but is also responsible for increased rates of severe drug abuse and addiction owing to its characteristic psychoactive effect. Non-medical illicit use of the poppy plant is markedly increasing worldwide, thereby highlighting the need for a robust species identification strategy. In this study, we identified SNPs within the region of two universal DNA barcodes, matK (maturase K) and the trnL-trnF (tRNA-Leu [3′exon]-tRNA-Phe [exon] intergenic spacer, that are forensically applicable for distinguishing opium poppy species based on a genetic analysis of 164 samples of family Papaveraceae obtained from locations spanning Jeolla-do and Jeju Island, Republic of Korea. A comparative analysis of the DNA barcode sequences for two narcotic types of the Papaver species (Papaver somniferum, Papaver somniferum subs. setigerum) to eight non-narcotic species revealed three unique nucleotide substitution events. Newly identified SNPs were located at position 255 of matK and at positions 305 and 306 of trnL-trnF; the narcotic species contained C, A, and T, whereas non-narcotic species contained T, G, and C at these positions. Phylogenetic analysis demonstrated that newly identified SNPs, which we named PsMAT255 and PsLF305/306, could be used to clearly differentiate between the narcotic and non-narcotic types of Papaver species based on the patterns of nucleotide variation. These results indicate that the nucleotide differences between the narcotic and non-narcotic species may influence genetic markers. We, therefore, developed a novel SNP-based allelic genotyping assay using the RT-PCR system that can reliably differentiate the narcotic type of the Papaver species. In summary, our findings suggest that the newly identified species-specific SNPs of both matK and trnL-trnF can be used as identification markers of narcotic Papaver species. Furthermore, a newly developed TaqMan allelic discrimination assay may be used as a practically applicable diagnostic method to survey several illicit narcotic specimens carrying the type-specific SNP.

Covariance predicts conserved protein residue interactions important for the emergence and continued evolution of SARS-CoV-2 as a human pathogen.

SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that likely emerged from animal reservoirs. Differences in nucleotide and protein sequence composition within related β-coronaviruses are often used to better understand CoV evolution, host adaptation, and their emergence as human pathogens. Here we report the comprehensive analysis of amino acid residue changes that have occurred in lineage B β-coronaviruses that show covariance with each other. This analysis revealed patterns of covariance within conserved viral proteins that potentially define conserved interactions within and between core proteins encoded by SARS-CoV-2 related β-coronaviruses. We identified not only individual pairs but also networks of amino acid residues that exhibited statistically high frequencies of covariance with each other using an independent pair model followed by a tandem model approach. Using 149 different CoV genomes that vary in their relatedness, we identified networks of unique combinations of alleles that can be incrementally traced genome by genome within different phylogenic lineages. Remarkably, covariant residues and their respective regions most abundantly represented are implicated in the emergence of SARS-CoV-2 and are also enriched in dominant SARS-CoV-2 variants.

Internal transcribed spacer 2 (ITS2) based molecular identification of malaria vectors from Bangsring Banyuwangi-Indonesia

Since the malaria outbreak in 2011, the breeding place of Anopheles in Bangsring Village on Banyuwangi District has been monitored by District Public Health Office as part of a vector surveillance program. Morphological identification is still a standard tool to observe Anopheles occurrence and diversity, but the presence of cryptic species made it unreliable. In this study, a molecular approach called DNA barcoding technique was used to assist the morphology-based techniques to identify Anopheles species found in Bangsring. The internal transcribed spacer 2 (ITS2) sequence was used as molecular marker. Based on the morphological features, we were able to identify Anopheles (An.) vagus, An. subpictus, An. sundaicus and An. aconitus. ITS2 sequences from the four identified species were then analyzed simultaneously with eighteen reference sequences from NCBI which had a high similarity of 98-100%. The NJ phylogenetic tree formed three major clades, where the two clades as monophyletic clades were An. vagus and An. aconitus. Another clade was formed as polyphyletic clade containing An. subpictus and An. sundaicus. Although An. subpictus and An. sundaicus were placed in the same clade, seven nucleotide differences were observed in their ITS2 sequence. The intra-specific variation of those two species was 0.08 and 0.49%, respectively, while the interspecific variation was 1.39%. Interspecific variation which was higher than the mean intra-specific variation might indicate that An. sundaicus and An. subpictus were a distantly species. However, the value of interspecific variation lower than 3% might also indicate that those species were classified as a complex species. All ITS2 sequences from morphologically identified species had similar results with molecular-based techniques. This result showed that molecular identification using the ITS2 sequence was reliable in supporting morphological identification among closely related anopheline mosquitoes and gave further information about their evolutionary divergence.

Wickerhamomyces sinyiensis f.a., sp. nov., a new ascomycetous yeast species in the Wickerhamomyces clade isolated in Taiwan.

This study describes Wickerhamomyces sinyiensis, a new anamorphic ascomycetous yeast species, four strains of which were isolated from soil and the fruiting body of a mushroom in Taiwan between 2006 and 2007. Analysis of the sequences of the large-subunit rRNA, small-subunit rRNA and elongation factor-1α identified this species as a member of the Wickerhamomyces clade. The yeast strains of W. sinyiensis exhibited a 0-3 nucleotide difference in the sequences of the D1/D2 domain of the large subunit rRNA when compared to one another and a 10 and 11 nucleotide difference when compared to Candida sp. BG99-11-14-10-4-1 and NRRL Y-7574, the closest undescribed species, respectively. The yeast strains differed by 77 and 78 nucleotides from W. orientalis and W. bispora, the close Wickerhamomyces species, respectively. The internal transcribed spacer sequences of the four isolates exhibited a divergence of 106-108 substitutions from the recognized species W. xylosivorus. No sexual reproduction was observed. The strains differed from those of related species in terms of their carbon and nitrogen assimilation patterns. Therefore, this study proposes W. sinyiensis f.a., sp. nov. to accommodate these four strains, with W. sinyiensis BCRC 23185T (isotype CBS 11432T; MycoBank number MB563484) as the holotype.

Programmable, Universal DNAzyme Amplifier Supporting Pancreatic Cancer-Related miRNAs Detection

The abnormal expression of miRNA is closely related to the occurrence of pancreatic cancer. Herein, a programmable DNAzyme amplifier for the universal detection of pancreatic cancer-related miRNAs was proposed based on its programmability through the rational design of sequences. The fluorescence signal recovery of the DNAzyme amplifier showed a good linear relationship with the concentration of miR-10b in the range of 10–60 nM, with a detection limit of 893 pM. At the same time, this method displayed a high selectivity for miR-10b, with a remarkable discrimination of a single nucleotide difference. Furthermore, this method was also successfully used to detect miR-21 in the range of 10–60 nM based on the programmability of the DNA amplifier, exhibiting the universal application feasibility of this design. Overall, the proposed programmable DNAzyme cycle amplifier strategy shows promising potential for the simple, rapid, and universal detection of pancreatic cancer-related miRNAs, which is significant for improving the accuracy of pancreatic cancer diagnosis.

Chromosome-level assembly and annotation of the blue catfish Ictalurus furcatus, an aquaculture species for hybrid catfish reproduction, epigenetics, and heterosis studies.

BackgroundThe blue catfish is of great value in aquaculture and recreational fisheries. The F1 hybrids of female channel catfish (Ictalurus punctatus) × male blue catfish (Ictalurusfurcatus) have been the primary driver of US catfish production in recent years because of superior growth, survival, and carcass yield. The channel–blue hybrid also provides an excellent model to investigate molecular mechanisms of environment-dependent heterosis. However, transcriptome and methylome studies suffered from low alignment rates to the channel catfish genome due to divergence, and the genome resources for blue catfish are not publicly available.ResultsThe blue catfish genome assembly is 841.86 Mbp in length with excellent continuity (8.6 Mbp contig N50, 28.2 Mbp scaffold N50) and completeness (98.6% Eukaryota and 97.0% Actinopterygii BUSCO). A total of 30,971 protein-coding genes were predicted, of which 21,781 were supported by RNA sequencing evidence. Phylogenomic analyses revealed that it diverged from channel catfish approximately 9 million years ago with 15.7 million fixed nucleotide differences. The within-species single-nucleotide polymorphism (SNP) density is 0.32% between the most aquaculturally important blue catfish strains (D&B and Rio Grande). Gene family analysis discovered significant expansion of immune-related families in the blue catfish lineage, which may contribute to disease resistance in blue catfish.ConclusionsWe reported the first high-quality, chromosome-level assembly of the blue catfish genome, which provides the necessary genomic tool kit for transcriptome and methylome analysis, SNP discovery and marker-assisted selection, gene editing and genome engineering, and reproductive enhancement of the blue catfish and hybrid catfish.

Rapid and Affordable High Throughput Screening of SARS-CoV-2 Variants Using Denaturing High-Performance Liquid Chromatography Analysis

Mutations in the receptor binding domain (RBD) of SARS-CoV-2 alter the infectivity, pathogenicity, and transmissibility of new variants of concern (VOCs). In addition, those mutations cause immune escape, undermining the population immunity induced by ongoing mass vaccination programs. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOCs. The aim of this study was to provide a quick, cheap and straightforward denaturing high-performance liquid chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOCs. Two PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably form whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect all the variants including B.1.1.7, P.1, B.1.585 B.1. 617.2 and lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants, even in the case of samples with very unbalanced target concentrations including those samples at the limit of detection. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants.

Genetic characterization of Salmonella Infantis from South Africa, 2004-2016.

Salmonella Infantis is presenting an increasing risk to public health. Of particular concern are the reports of pESI, a multidrug resistance (MDR) encoding megaplasmid, in isolates from multiple countries, but little is known about its presence or diversity in South Africa. Whole genome sequences of 387 S. Infantis isolates from South Africa (2004–2020) were analysed for genetic phylogeny, recombination frequency, antimicrobial resistance (AMR) determinants, plasmid presence and overall gene content. The population structure of South African S. Infantis was substantially different to S. Infantis reported elsewhere; only two thirds of isolates belonged to eBG31, while the remainder were identified as eBG297, a much rarer group globally. Significantly higher levels of recombination were observed in the eBG297 isolates, which was associated with the presence of prophages. The majority of isolates were putatively susceptible to antimicrobials (335/387) and lacked any plasmids (311/387); the megaplasmid pESI was present in just one isolate. A larger proportion of eBG31 isolates, 19% (49/263), contained at least one AMR determinant, compared to eBG297 at 2% (3/124). Comparison of the pan-genomes of isolates from either eBG identified 943 genes significantly associated with eBG, with 43 found exclusively in eBG31 isolates and 34 in eBG297 isolates. This, along with the single nucleotide polymorphism distance and difference in resistance profiles, suggests that eBG31 and eBG297 isolates occupy different niches within South Africa. If antibiotic-resistant S. Infantis emerges in South Africa, probably through the spread of the pESI plasmid, treatment of this infection would be compromised.

Genetic Diversity and Gene Flow Observed in Two Cereal Aphid (Hemiptera: Aphididae) Species and Populations in the Chinese Corn Belt Region

Abstract The cereal aphids, Rhopalosiphum maidis (Fitch) and Rhopalosiphum padi (L.) (Hemiptera: Aphididae), are serious pests of maize, Zea mays L., globally. Basic information about the genetic structure of these aphids is unknown. We, therefore, estimated the genetic diversity and genetic flow from partial fragments of the mitochondrial cytochrome oxidase I gene collected from different geographic populations of aphids throughout the Songliao Plain of northeastern China. Our analysis of a 425-bp sequence on 297 R. maidis and 287 R. padi individuals revealed 17 and 3 variable sites, respectively. Fifteen haplotypes were obtained among the R. maidis populations, and only haplotype RM1 was shared. Three shared haplotypes were obtained among the R. padi populations. No obvious geographical trends were detected based on the haplotype network and neighbor-joining tree. Relatively low haplotype diversity indices were observed in the R. maidis populations (haplotype diversity [Hd] = 0.14207, average number of nucleotide differences [K] = 0.17340, and nucleotide diversity [Pi] = 0.00041), whereas relatively high haplotype diversity indices were observed in the R. padi populations (Hd = 0.53249, K = 1.46614, and Pi = 0.00345). There was moderate gene flow (number of migrants [Nm] = 2.33) among R. maidis populations, but there was low gene flow (Nm = 0.82) among R. padi populations. Analysis of molecular variance showed high genetic differentiation within populations. Genetic distance and geographic distance were not significantly associated according to the Mantel test. The results suggest that the difference in the aphids' existence strategies has resulted in a different mitochondrial evolution pattern in the Songliao Plain region, and they provide a foundation for accurately forecasting systems against this pest.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis.

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.

Characterization of infectious laryngotracheitis virus isolates from laying hens during 2019-2020 outbreaks in Tamil Nadu, India.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India.

Complete genome sequence of a novel virus belonging to the genus Badnavirus in jujube (Ziziphus jujuba Mill.) in China.

The genome sequence of a novel circular DNA virus related to members of the genus Badnavirus was identified in diseased jujube trees by high-throughput sequencing and verified by conventional Sanger sequencing of cloned PCR products. The name "jujube badnavirus WS" (JuBWS) is proposed for this virus. Diseased jujube leaves showed yellow mosaic and malformation symptoms, with round chlorotic spots found on diseased fruit. The genome of this virus has a length of 6450 nt and has a typical badnavirus genomic structure with three open reading frames (ORFs). JuBWS was identified as a novel badnavirus based on nucleotide differences in the RNase (RT + RNase H) coding region of ORF3. The JuBWS sequence showed 70.48-76.41% nucleotide sequence identity to other known badnaviruses, thus meeting the taxonomic criterion for establishing a new species within the genus Badnavirus. This study suggested that the novel badnavirus might be a pathogen associated with jujube mosaic disease, and this will be investigated in the future.

Assessment of new sequencing information for genetically modified cotton DAS-24236-5 × DAS-21Ø23-5.

The GMO Panel has previously assessed genetically modified (GM) cotton DAS‐24236‐5 × DAS‐21Ø23‐5 and concluded that it is as safe as its conventional counterpart and other appropriate comparators with respect to potential effects on human and animal health and the environment in the context of its intended uses. On 17 November 2020, the European Commission requested EFSA to evaluate new DNA sequence information and updated bioinformatics data for cotton DAS‐24236‐5 × DAS‐21Ø23‐5 and to indicate whether the conclusions of the GMO Panel on the previously assessed cotton DAS‐24236‐5 × DAS‐21Ø23‐5 remain valid. The new sequence data of DAS‐24236‐5 showed the change of one nucleotide that results in one amino acid substitution, in the newly expressed Cry1F (synpro_L620Q) compared to the sequence originally reported. The GMO Panel concludes that this amino acid substitution in the protein is a mutation. Nonetheless with the exception of the bioinformatics analysis, the studies performed for the risk assessment of Cry1F in cotton DAS‐24236‐5 × DAS‐21Ø23‐5 remain valid. In addition, the new sequencing data showed a change in one nucleotide in the 5′ flanking region of DAS‐21Ø23‐5 compared to the original sequence reported on the stack cotton DAS‐24236‐5 × DAS‐21Ø23‐5. The bioinformatic analyses of the newly sequenced DAS‐21Ø23‐5 event in the stack DAS‐24236‐5 × DAS‐21Ø23‐5 shows that the nucleotide difference is in the 5′ flanking region outside the ORFs that span the 5′ junction and is therefore not considered further in the safety assessment. Based on the information provided, the GMO Panel concludes that the corrected sequence does not give rise to any safety concerns, and therefore, the original risk assessment of cotton DAS‐24236‐5 × DAS‐21Ø23‐5 remains valid.

Risk assessment of new sequencing information for genetically modified sugar beet H7-1.

The EFSA Panel on genetically modified organisms (GMO Panel) has previously assessed genetically modified (GM) sugar beet H7‐1. This sugar beet was found to be as safe and nutritious as its conventional counterpart and commercial sugar beet varieties with respect to potential effects on human and animal health and the environment in the context of its intended uses. On 19 February 2021, European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for GM sugar beet H7‐1 and to indicate whether the previous conclusions of the GMO Panel on safety of GM sugar beet H7‐1 remain valid. The new sequencing data indicated seven nucleotide differences as compared to the sequence originally provided in applications EFSA‐GMO‐UK‐2004‐08 and EFSA‐GMO‐RX‐006: five nucleotides in the 5’ genomic flanking region and two nucleotides in the T‐DNA region of the insert. Two mismatches between the originally submitted H7‐1 event sequence and the plasmid sequence were confirmed by the newly obtained H7‐1 sequence. Based on the analysis of the provided data, EFSA considers that the newly reported sequence differences are most likely attributed to sequencing errors in the originally reported sugar beet H7‐1 event sequence. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of sugar beet H7‐1 remains valid.

Nucleotide sequence types (ntSTs) of Anaplasma marginale in cattle in Nigeria based on the major surface protein 5 (msp5) gene

Bovine anaplasmosis caused by Anaplasma marginale is an important endemic disease that exerts negative impact on livestock production with huge socioeconomic consequences in most developing countries. Genetic studies have reported the existence of diverse ntSTs of A. marginale with varying pathogenicity in different countries. Continuous studies to obtain accurate information on disease etiologies is desirable for the formulation of cost-effective control measures. To this end, 582 blood samples from cattle were collected from 10 out of the 36 States of Nigeria from April 2021 to March 2022 and analyzed based on the major surface protein 5 (msp5) gene to determine the ntSTs of A. marginale in Nigeria. In all, 38 out of the 582 samples (6.5%) from cattle in the different Agro-ecological Zones (AEZs) of Nigeria were positive. The Nigerian A. marginale nucleotide sequences were 96.7 to 100% identical to sequences from other countries and were placed in distinct clusters with other A. marginale sequences deposited in GenBank. Network analysis revealed three ntSTs (#2, #4 & #8) of A. marginale from Nigeria with a nucleotide sequence type diversity (Hd) of 0.8, nucleotide diversity (Pi) of 0.015 and average number of nucleotide differences (k) of 7.09. Two different amino acid substitution sites were found in Nigerian and worldwide sequences at positions 148 and 160. This is the first nationwide report on the ntST diversity and genetic characterization of A. marginale in cattle in Nigeria based on the msp5 gene. Bovine anaplasmosis is widespread in Nigeria and deserves further attention.

Genetic Diversity among Four Populations of Aedes aegypti (Diptera: Culicidae) from Honduras as Revealed by Mitochondrial DNA Cytochrome Oxidase I.

Aedes aegypti is a hematophagous and highly anthropophilic mosquito with a wide distribution, particularly in tropical and subtropical regions of the world. Ae. aegypti is the main vector of several febrile diseases called arboviruses (dengue, yellow fever, chikungunya, and zika viruses), which represent an important public health problem. Populations of this mosquito were nearly eliminated from the Americas in the mid-20th century; however, after the abandonment of control measures, mosquito populations have been recovering territory, have expanded by anthropogenic mechanisms, and have been joined by new populations reintroduced from other continents. The objective of this pilot study was to determine the genetic variability of Aedes aegypti collected in four cities located along the so-called logistics corridor of Honduras, which connects the Caribbean Sea to the Pacific Ocean. We studied the sequences of two molecular markers: the cytochrome c oxidase 1 (COI mtDNA) gene and the internal transcribed spacer 2 (ITS2 rDNA) of 40 mosquitoes. Phylogenetic analyzes show two separate clades with a low number of nucleotide differences per site, three haplotypes, and low haplotype diversity. These results suggest a low genetic diversity in the populations of Ae. aegypti in Honduras in relation to that reported in other countries of the Central American isthmus.