A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

Heterodera amaranthusiae n. sp. (Nematoda: Heteroderidae), a new cyst nematode parasitising Amaranthus retroflexus L. in China

Summary A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus, in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μm, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μm with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μm long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, AluI, BsuRI (HaeIII) and CfoI (HhaI), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.

A simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following three groups: high methylation (HM), intermediate methylation (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML have been analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n=99) using the DREAM data has identified HM and LM subgroups (HM_DREAM, n=35; LM_DREAM; n=64). Of the 98 cases that could be compared with the international consensus classification, 90 cases of HM (n=30) and LM (n=60) had 100% concordance with the DREAM clustering results. For the remaining eight cases classified as the IM group, four cases were classified into the HM_DREAM group and four cases into the LM_DREAM group. A machine-learning classifier has been successfully constructed using a Support Vector Machine (SVM), which divided the validation cohort (n=38) into HM (HM_SVM; n=18) and LM (LM_SVM; n=20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, a robust methylation test has been developed using the DREAM analysis for patients with JMML. This simple and straightforward test can be easily incorporated in diagnosis to generate a methylation classification for patients so that they can receive risk-adapted treatment in the context of future clinical trials.

A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.

Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia.

Importance of mutations in amino acid 484 of the Spike protein of SARS-CoV-2: rapid detection by restriction enzyme analysis

Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 484 of the Spike protein are particularly important and associated with some of these variants: E484K or E484Q. These mutations have been associated with evasion to neutralizing antibodies. Restriction enzyme analysis is proposed as a rapid method to detect these mutations. A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. E484K, present in some VOCs, has emerged in several other lineages and is frequently found in recent viral isolates. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, and MseI. The use of these two enzymes allows the detection of mutations at position 484, and to differentiate between these three conditions: non-mutated, and the presence of E484K or E484Q. A 100% correlation was observed with sequencing results. The proposed methodology, which allows for the screening of a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.

EXPRESSION OF CELLULOSE-DEGRADING ENDOGLUCANASE FROM BACILLUS SUBTILIS USING PTOLT EXPRESSION SYSTEM IN ESCHERICHIA COLI

Endoglucanases randomly hydrolyse the cellulose chains by acting upon internal β-1,4-D-glycosidic bonds and are used extensively in industrial applications. In this study, bacterial endoglucanase gene yhfE was obtained by PCR, using primers based on genomic sequences of Bacillus subtilis strains. 1041 bp DNA fragment of yhfE was cloned into Escherichia coli DH5α through the use of pTolT expression plasmid. PCR, restriction enzyme analysis and DNA sequencing were performed in order to confirm the cloning. E. coli BL21-AI cells expressed the yhfE after induction at 0.04% of arabinose concentration for 4 h. The expected 38.7 kDa size yhfE protein after digestion with thrombin of the His-tagged fusion protein (yhfE-TolAIII) was visualized by SDS-PAGE. The yhfE-TolAIII production yield was approximately 82 mg/L. The recombinant yhfE was characterized by MALDI-TOF mass spectrometry and CD analysis.

Fatal Neonatal Sepsis Associated with Human Adenovirus Type 56 Infection: Genomic Analysis of Three Recent Cases Detected in the United States

Background: Human adenovirus (HAdV)-D56 was first described in 2011 by genomics analysis of a strain isolated in France in 2008 from a fatal case of neonatal infection. Since then, it has been reported in cases of keratoconjunctivitis and male urethritis. Three epidemiologically unrelated fatal cases of neonatal sepsis associated with infection by HAdV-D strains with a similar genetic makeup were documented in the United States between 2014 and 2020. Methods: Whole genome sequences were obtained for the isolated strains, and genomics analyses were conducted to compare them to phylogenetically related HAdV-D genomic sequences available in GenBank. Results: The three new US strains were indistinguishable by in silico restriction enzyme analysis. Their genome sequences were 99.9% identical to one another and to the prototype strain isolated in 2008 from a similar context of disease. The phylogenetic reconstruction revealed a highly supported clustering of all HAdV-D56 strains isolated in various countries since 1982. Our comparison to serologically intermediate strains 15/H9 described in the literature indicated that HAdV-D56-like viruses have circulated worldwide since the late 1950s. Conclusion: As with other HAdV-D genotypes with the ability to infect ocular and genital mucosae, the risk of severe prenatal or perinatal HAdV-D56 infection must be considered.

Whole human genome 5'-mC methylation analysis using long read nanopore sequencing

DNA methylation is a type of epigenetic modification that affects gene expression regulation and is associated with several human diseases. Microarray and short read sequencing technologies are often used to study 5'-methylcytosine (5'-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustable results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulfite and the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5'-mC since the long reads produced by this technology allow to resolve those genomic regions more easily. Moreover, it allows direct sequencing of native DNA molecules using a fast library preparation procedure. In this work we show that 10X coverage depth nanopore sequencing, using DNA from a human cell line, produces 5'-mC methylation frequencies consistent with those obtained by methylation microarray and digital restriction enzyme analysis of methylation. In particular, the correlation of methylation values ranged from 0.73 to 0.90 using an average genome sequencing coverage depth <2X or a minimum read support of 17X for each CpG site, respectively. We also showed that a minimum of 5 reads per CpG yields strong correlations (>0.89) between sequencing runs and an almost uniform variation in methylation frequencies of CpGs across the entire value range. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns according to genomic annotations, including a majority of unmethylated and methylated sites in the CpG islands and inter-CpG island regions, respectively. These results demonstrate that low coverage depth nanopore sequencing is a fast, reliable and unbiased approach to the study of 5'-mC in the human genome.

Importance of E484K and N501Y mutations in SARS-CoV-2 for genomic surveillance: rapid detection by restriction enzyme analysis

Introduction: Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus responsible for COVID-19, have emerged in several countries. Two mutations in the gene coding for the Spike protein of the viral genome are particularly important and associated with some of these variants: E484K and N501Y. Restriction enzyme analysis is proposed as a rapid method to detect these two mutations. Methodology: A search on GISAID was performed in April 2021 to detect the frequency of these two mutations in the sequence available and their association with other lineages. A small amplicon from the Spike gene was digested with two enzymes: HpyAV, which allows detecting E484K mutation, and MseI, for detecting the N501Y one. Results: The mutations E484K and N501Y, associated with VOCs, have emerged in several other lineages, particularly E484K. A 100% correlation was observed with sequencing results. Conclusions: The proposed methodology, which allows screening a great number of samples, will probably help to provide more information on the prevalence and epidemiology of these mutations worldwide, to select the candidates for whole-genome sequencing.