Complete Genome Sequence and Phylogenetic Analysis of a Goose Astrovirus Isolate in China

Astroviruses are considered the cause of gastroenteritis in humans and animals. Studies in recent years show avian astroviruses are also associated with duckling hepatitis, gosling gout, and chicken nephritis. In this study, a GAstV strain, designated as JS2019/China, was detected in dead goslings from a commercial goose farm in Jiangsu province of China. Viral strain was proliferated in goose embryos and sequence analysis showed the isolated strain had a classical structure arrangement and a series of conserved regions compared with other GAstVs. Sequence comparison and phylogenetic analysis of whole genome and ORF2 revealed that JS2019/China belongs to the GAstV group-1, which consists of most of the GAstV strains. Amino acid analysis indicated that some mutants might have an impact on viral protease capacity, such as V505I and K736E of ORF1a and T107I, F342S, and S606P of ORF2. Taken together, a novel GAstV strain was isolated and genomic analysis and protein polymorphism analysis indicated that some amino acid mutants might affect the viral virulence.

A Novel Gene, Le-Dd 10, Is Involved in Fruiting Body Formation of Lentinula Edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened by using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd 1~21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

Polymorphism analysis of pfmdr1 gene in Plasmodium falciparum isolates 11 years post-adoption of artemisinin-based combination therapy in Saudi Arabia

A total of 227 Plasmodium falciparum isolates from Jazan region, southwestern Saudi Arabia were amplified for the P. falciparum multi-drug resistance 1 (pfmdr1) gene to detect point mutations 11 years after the introduction of artemisinin-based combination therapy (ACT) in Saudi Arabia. The pfmdr1 86Y mutation was found in 11.5% (26/227) of the isolates while the N86 wild allele was detected in 88.5%. Moreover, 184F point mutations dominated (86.3%) the instances of pfmdr1 polymorphism while no mutation was observed at codons 1034, 1042 and 1246. Three pfmdr1 haplotypes were identified, NFSND (74.9%), NYSND (13.7%) and YFSND (11.4%). Associations of the prevalence of 86Y mutation and YFSND haplotype with participants’ nationality, residency and parasitaemia level were found to be significant (P < 0.05). The findings revealed significant decline in the prevalence of the pfmdr1 86Y mutation in P. falciparum isolates from Jazan region over a decade after the implementation of ACT treatment. Moreover, the high prevalence of the NFSND haplotype might be indicative of the potential emergence of CQ-sensitive but artemether-lumefantrine-resistant P. falciparum strains since the adoption of ACT. Therefore, continuous monitoring of the molecular markers of antimalarial drug resistance in Jazan region is highly recommended.

Genetic Characterization of mcr-1-Positive Multidrug-Resistant Salmonella enterica Serotype Typhimurium Isolated From Intestinal Infection in Children and Pork Offal in China

With the rapid emergence of plasmid-mediated colistin resistance gene mcr-1, the increased resistance of Salmonella has attracted extensive attention. This study reports on 11 multidrug-resistant Salmonella enterica serovar Typhimurium strains harboring mcr-1 in China. They all presented resistance to colistin, and additionally, one that was isolated from a child’s stool sample was also resistant to ceftriaxone and azithromycin. We screened 1454 strains of Salmonella for mcr-1 gene through PCR, and these strains are all preserved in our laboratory. Antimicrobial sensitivity analysis was carried out for the screened mcr-1 positive strains. Genetic polymorphism analysis of S. Typhimurium was performed by using the Pulsed-Field Gel Electrophoresis (PFGE). The plasmids harboring mcr-1 were identified by S1-PFGE and southern blotting. Plasmid conjugation assays were used to analyze the transferability of colistin resistance. The plasmids harboring mcr-1 were characterized by sequencing and bioinformatic analysis. Eleven S. Typhimurium strains harboring mcr-1 with colistin resistance (MICs 4μg/ml) were detected, which were isolated from children and pig offal in China. All of them were multidrug-resistant strains. PFGE results revealed that the strains isolated from different samples or locations have identical genotypes. S1-PFGE and southern blotting experiments showed that three plasmids of different sizes (33, 60, and 250 kb) all carried the mcr-1 gene. The plasmid conjugation assays revealed that Salmonella acquired mcr-1 harboring plasmids by horizontal transfer. Sequencing and plasmid type analysis revealed that these plasmids were types IncX4, IncI2, and IncHI2. Among them, IncX4 and IncI2 plasmids had extremely similar backbones and contained one resistant gene mcr-1. IncHI2 plasmid contained multiple resistant genes including blaCTX–M, oqxB, sul, aph, aadA, and blaTEM. We identified 11 mcr-1 harboring S. Typhimurium strains in China and described their characteristics. Our findings indicate that the mcr-1 gene can effectively spread among intestinal bacteria by horizontal transfer of three types of plasmids. Moreover, the IncHI2 plasmid can also mediate the transfer of other drug resistance genes. These results reveal that constant surveillance of mcr-1 harboring S Typhimurium is imperative to prevent the spread of colistin resistance.

Synaptosomal-Associated Protein 25 Gene Polymorphisms Affect Treatment Efficiency of Methylphenidate in Children With Attention-Deficit Hyperactivity Disorder: An fNIRS Study

Methylphenidate (MPH) is the first-line drug for the treatment of children with attention-deficit hyperactivity disorder (ADHD); however, individual curative effects of MPH vary. Many studies have demonstrated that synaptosomal-associated protein 25 (SNAP-25) gene MnlI polymorphisms may be related to the efficacy of MPH. However, the association between SNAP-25MnlI polymorphisms and changes in brain hemodynamic responses after MPH treatment is still unclear. This study used functional near-infrared spectroscopy (fNIRS) to preliminarily investigate the interaction of MPH treatment-related prefrontal inhibitory functional changes with the genotype status of the SNAP-25 gene in children with ADHD. In total, 38 children with ADHD aged 6.76–12.08 years were enrolled in this study and divided into the following two groups based on SNAP-25 gene MnlI polymorphisms: T/T genotype group (wild-type group, 27 children) and G allele carrier group (mutation group, 11 children). The averaged oxygenated hemoglobin concentration changes [Δavg oxy-Hb] and deoxyhemoglobin concentration changes [Δavg deoxy-Hb] in the frontal cortex before MPH treatment and after 1.5 h (post-MPH1.5h) and 4 weeks (post-MPH4w) of MPH treatments were monitored using fNIRS during the go/no-go task. SNAP-IV scores were evaluated both pre-MPH and post-MPH4w treatments. In the T/T genotype group, [Δavg oxy-Hb] in the dorsolateral prefrontal cortex was significantly higher after 4 weeks of MPH (post-MPH4W) treatment than pre-treatment; however, in the G allele group, no significant differences in [Δavg oxy-Hb] were observed between pre- and post-treatments. In the go/no-go task, the accuracy was significantly increased post-MPH4w treatment in the T/T genotype group, while no significant differences were observed in response time and accuracy of the “go” sand no-go task in the G allele group for pre-MPH, post-MPH1.5h, and post-MPH4w treatments. The T/T genotype group exhibited a significant decrease in SNAP-IV scores after MPH treatment, while the G allele group showed no significant difference. In conclusion, fNIRS data combined with SNAP-25 MnlI polymorphism analysis may be a useful biomarker for evaluating the effects of MPH in children with ADHD.

Single Nucleotide Polymorphism Analysis of Pvmdr-1 in Plasmodium Vivax Isolated from Military Personnel of Republic of Korea in 2016 and 2017

Background: Malaria chemoprophylaxis using chloroquine (CQ) and primaquine (PQ) has been administered to resident soldiers in the 3rd Army of Republic of Korea (ROK) to prevent malaria infection since the year 1997. Due to mass chemoprophylaxis against malaria, concern exists about occurrence of chloroquine resistance (CQR). Herein, we investigated the single nucleotide polymorphisms (SNPs) of the Plasmodium vivax multi-drug resistance protein-1 (pvmdr-1) gene to monitor the risk of CQR. Methods: SNPs of the pvmdr-1 gene were analyzed in 73 soldiers of the 3rd Army of ROK diagnosed with infection by Plasmodium vivax (P. vivax). Results: Quintuple mutations (G698S, L845F, M908L, T958M, and F1076L) were detected in 73 soldiers. Mutation in the Y541C position was firstly detected in soldiers at a frequency of 1.3% (1/73). In addition, synonymous mutations were detected at positions K44, L493, T529, and E1233. Based on these SNPs, pvmdr-1 sequences of ROK were classified into 6 haplotypes. The phylogenetic analysis closed to Type of North Korean showed that P. vivax malaria of ROK could be a reason of influx from North Korea. In this study, there was no therapeutic resistance (CQ-mediated parasite clearance within 72 hours) for clinical samples that possessed various SNPs of pvmdr-1. Various SNPs including a newly identified non-synonymous mutation (Y541C) had been introduced into P. vivax malaria-endemic areas in ROK. Conclusions: Our study showed that synonymous and non-synonymous mutations of pvmdr-1 were introduced to the malaria chemoprophylaxis-executed regions of ROK from 2016 to 2017. Thus, to prevent the emergence of CQR, continuous surveillance for SNPs of pvmdr-1 related with CQR in the malaria-endemic regions of ROK is essential.

VEGF and eNOS genes polymorphism features in patients with diabetes mellitus with and without initial non-proliferative diabetic retinopathy

The endothelial NO synthase (eNOS) and vascular endothelial growth factor (VEGF) imbalance and the polymorphism of these genes may be the predisposition for diabetic retinopathy (DR) development and progression.The aim: to analyze VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes polymorphism and their combinations in patients with type 2 diabetes mellitus (DM2) with and without initial non-proliferative DR.Materials and methods. The study included 200 patients with type 2 diabetes (155 women and 45 men, age – 43–70 years): 111 people without and 89 people with DR. The polymorphism of the regulatory regions of VEGF (rs699947 and rs3025039) and eNOS (rs2070744) genes was studied using restriction fragment length polymorphism analysis and TaqMan Real-Time PCR by. Statistical processing was carried out using the software packages Statistica 10.0, SPSS Statistics 23 and the package of original programs for volumetric processing of bioinformation.Results. The VEGF-2578 heterozygosity and two complex genotypes – VEGF-2578CA:VEGF+936CC and NOS3-786CT:VEGF-2578CA:VEGF+936CC – signifi cantly decreased in patients with DR. The predisposition to early DR development to minor genotype of eNOS gene in the NOS3-786CC:VEGF+936CT complex and signifi cantly decreased the homozygous wild-type eNOS genotype in DM2 patients with ophthalmopathology were shown. NOS3-86TT:VEGF2578AA genotype signifi cantly decreased in group with retinopathy developing and the glycated hemoglobin high level.Conclusion. Along with the clinical risk factors for the development of DR in DM2, the genetic polymorphism of the regulatory regions of the genes analyzed by us has a signifi cant weight. When analyzing potential genetic markers, it is important to consider possible joint epistatic/hypostatic effects. The complex analysis of polymorphic gene can help early prognosis of the DR development.

Assessment of the level of allelic polymorphism of SSR markers and genetic distances of some grape varieties of the South of Russia of different ecological-geographical groups

Представлены результаты оценки генетического разнообразия 24 местных сортов юга России, поддерживаемых на ампелографической коллекции ФГБУН «ВННИИВиВ «Магарач». ДНК-типирование сортов и оценка аллельного разнообразия выполнено с использованием 9 ядерных (nSSR) и 3 хлоропластных (cpSSR) микросателлитных локусов. Уровень полиморфизма nSSR локусов составил 100 %. Всего было идентифицировано 73 аллеля, в среднем 9.1 аллеля /локус. Минимальное количество аллелей идентифицировано в локусах ssrVrZAG64 и ssrZag83. Наибольшее количество аллелей выявлено в локусе ssrVvUCH29 (13 аллелей), диапазон размера которых составил 203 п.н. - 309 п.н. В результате анализа полиморфизма сpSSR-локусов идентифицировано 4 хлоротипа: А, В, С, D. Наиболее распростанен в группе изученных сортов хлоротип D (58 %). В статье обсуждается происхождение сортов на основе анализа их гаплотипов. По результатами анализа аллельного полиморфизма nSSR-локусов рассчитана матрица генетических дистанций, значения которой находились в диапазоне 0,33-0,94, построена дендрограмма, отражающая взаимоотношения между образцами. По степени генетического сходства выделились 3 основных кластера, в которых наблюдалась дифференциация или тенденция к дифференциации по эколого-географическим группам. The assessment results of genetic diversity of 24 local varieties of the South of Russia, maintained in the ampelographic collection of the FSBSI Institute Magarach are presented. DNA typing of cultivars and assessment of allelic diversity was performed using 9 nuclear (nSSR) and 3 chloroplast (cpSSR) microsatellite loci. The level of polymorphism of nSSR loci was 100%. A total of 73 alleles were identified with an average of 9.1 alleles per locus. The minimal number of alleles was observed in the ssrVrZAG64 and ssrZag83 loci. The biggest number of alleles was found in the ssrVvUCH29 locus (13 alleles), the size range of which was 203 bp-309 bp. As a result of polymorphism analysis of cpSSR loci, 4 chlorotypes were identified: A, B, C, D. Chlorotype D is the most widespread in the group of the studied cultivars (58%). The article discusses the origin of varieties based on the analysis of their haplotypes. Based on the results of the analysis of allelic polymorphism of nSSR loci, a matrix of genetic distances was calculated, the values of which were in the range of 0.33-0.94, and a dendrogram, reflecting the relationship between the samples, was constructed. According to the degree of genetic similarity, 3 main clusters were distinguished, in which differentiation or a tendency towards differentiation by ecological-geographical groups was observed.

Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

Identification of the Yield Traits Related Haplotype Combinations of Transcription Factor Genes TaHDZ34 in Common Wheat

Improvement of yield-traits is one of the predominating objectives in wheat breeding. Homeodomain-leucine zipper (HD-ZIP) transcription factor plays significant roles in plant growth and development. The TaHDZ34 (A, B and D sub-genomics) genes consisting of three members of the HD-ZIP IV transcription factor gene subfamily in wheat (Triticum aestivum L.) were cloned. Two haplotypes of TaHDZ34-7A, TaHDZ34-7B or TaHDZ34-7D were respectively identified after the sequence polymorphism analysis, and three functional molecular markers were developed. The TaHDZ34 genes were divided into eight haplotype combinations. Association analysis and distinct population validation jointly indicated that TaHDZ34 had the function of modulating grain number per spike, effective spikelet number per spike, 1,000 kernel weight, and flag leaf area per plant in wheat. Among all haplotype combinations of TaHDZ34, Hap-ABD was the most excellent one. Subcelluar localization showed that TaHDZ34-7A was localized in the nucleus. Interaction proteins of TaHDZ34-7A protein proved to be involved in protein synthesis/degradation, energy production and transportation, and photosynthesis processes. Geographic distribution and frequencies of TaHDZ34 haplotype combinations suggested that the Hap-Abd and Hap-AbD were preferential selection in Chinese wheat breeding programs. The high-yield related haplotype combinations Hap-ABD of TaHDZ34 provided beneficial genetic resources for marker-assisted selection of new wheat cultivars.