New data on the complete mitochondrial genome of Azygia robusta (Azygiidae) were obtained by the next-generation sequencing (NGS) approach. The mitochondrial DNA (mtDNA) of A. robusta had a length of 13 857 bp and included 12 protein-coding genes, two ribosomal genes, 22 transfer RNA genes, and two non-coding regions. The nucleotide sequences of the complete mitochondrial genomes of two A. robusta specimens differed from each other by 0.12 ± 0.03%. Six of 12 protein-coding genes demonstrated intraspecific variation. The difference between the nucleotide sequences of the complete mitochondrial genomes of A. robusta and Azygia hwangtsiyui was 26.95 ± 0.35%; the interspecific variation of protein-coding genes between A. robusta and A. hwangtsiyui ranged from 20.5 ± 0.9% (cox1) to 30.7 ± 1.2% (nad5). The observed gene arrangement in the mtDNA sequence of A. robusta was identical to that of A. hwangtsiyui. Codon usage and amino acid frequencies were highly similar between A. robusta and A. hwangtsiyui. The results of phylogenetic analyses based on mtDNA protein-coding regions showed that A. robusta is closely related to A. hwangtsiyui (belonging to the same suborder, Azygiida) that formed a distinct early-diverging branch relative to all other Digenea. A preliminary morphological analysis of paratypes of the two azygiid specimens studied showed visible morphological differences between them. The specimen extracted from Sakhalin taimen (Parahucho perryi) was most similar to A. robusta. Thus, we here provide the first record of a new definitive host, P. perryi, for A. robusta and also molecular characteristics of the trematode specimens.

During the last two decades, the world has witnessed the emergence and re-emergence of arthropod-borne viruses, better known as arboviruses. The close contact between sylvatic, rural and peri-urban vector species and humans has been mainly determined by the environment-modifying human activity. The resulting interactions have led to multiple dead-end host infections and have allowed sylvatic arboviruses to eventually adapt to new vectors and hosts, contributing to the establishment of urban transmission cycles of some viruses with enormous epidemiologic impact. The metagenomic next-generation sequencing (NGS) approach has allowed obtaining unbiased sequence information of millions of DNA and RNA molecules from clinical and environmental samples. Robust bioinformatics tools have enabled the assembly of individual sequence reads into contigs and scaffolds partially or completely representing the genomes of the microorganisms and viruses being present in biological samples of clinical relevance. In this review, we describe the different ecological scenarios for the emergence of viral diseases, the virus adaptation process required for the establishment of a new transmission cycle and the usefulness of NGS and computational methods for the discovery and routine genomic surveillance of mosquito-borne viruses in their ecosystems.

Although immune checkpoint inhibitors (ICIs) have produced remarkable responses in non-small cell lung cancer (NSCLC) patients, receivers still have a relatively low response rate. Initial response assessment by conventional imaging and evaluation criteria is often unable to identify whether patients can achieve durable clinical benefit from ICIs. Overall, there are sparse effective biomarkers identified to screen NSCLC patients responding to this therapy. A lot of studies have reported that patients with specific gene mutations may benefit from or resist to immunotherapy. However, the single gene mutation may be not effective enough to predict the benefit from immunotherapy for patients. With the advancement in sequencing technology, further studies indicate that many mutations often co-occur and suggest a drastic transformation of tumour microenvironment phenotype. Moreover, co-mutation events have been reported to synergise to activate or suppress signalling pathways of anti-tumour immune response, which also indicates a potential target for combining intervention. Thus, the different mutation profile (especially co-mutation) of patients may be an important concern for predicting or promoting the efficacy of ICIs. However, there is a lack of comprehensive knowledge of this field until now. Therefore, in this study, we reviewed and elaborated the value of cancer mutation profile in predicting the efficacy of immunotherapy and analysed the underlying mechanisms, to provide an alternative way for screening dominant groups, and thereby, optimising individualised therapy for NSCLC patients.

Blood parasites comprise some of the most prevalent pathogens in nature, and their detection and identification are major objectives in varied fields such as ecology and biomedicine. Two approaches were compared, one based on Sanger sequencing and the other next-generation sequencing (NGS) based, in terms of their performance in detecting avian blood parasites across tropical Southeast Asian birds. Across a panel of 528 bird individuals, 43 birds were ascertained to be infected with avian haemosporidians using a polymerase chain reaction-based detection method. Among these samples, NGS-based barcoding confirmed co-infections by multiple blood parasites in all eight cases where Sanger sequencing produced double peaks. Importantly however, the NGS-based method produced another five diagnoses of co-infections (62.5%) in which Sanger-based barcoding remained equivocal. In contrast to Sanger sequencing, the NGS-based method was able to identify co-infecting haemosporidian lineages via their barcodes. The accuracy of avian haemosporidian lineage identification was not compromised by the shorter length of NGS sequences, with ~94% of NGS barcodes producing matches identical to those of the Sanger barcodes. The application of NGS-based barcoding methods promises to enhance parasite identification and reduce erroneous inferences based on artefacts.

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks among health care workers have been scarcely reported so far. This report presents the results of an epidemiologic and molecular investigation of a SARS-CoV-2 outbreak among laundromat facility workers in a large tertiary centre in Israel. Following the first three reported cases of SARS-CoV-2 among laundromat workers, all 49 laundromat personnel were screened by qRT-PCR tests using naso- and oropharingeal swabs. Epidemiologic investigations included questionnaires, interviews and observations of the laundromat facility. Eleven viral RNA samples were then sequenced, and a phylogenetic analysis was performed using MEGAX.

The integrated investigation defined three genetic clusters and helped identify the index cases and the assumed routes of transmission. It was then deduced that shared commute and public showers played a role in SARS-CoV-2 transmission in this outbreak, in addition to improper PPE use and social gatherings (such as social eating and drinking). In this study, we present an integrated epidemiologic and molecular investigation may help detect the routes of SARS-CoV-2 transmission, emphasising such routes that are less frequently discussed. Our work reinforces the notion that person-to-person transmission is more likely to cause infections than environmental contamination (e.g. from handling dirty laundry).

We report a familial cluster of 24 individuals infected with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The index case had a travel history and spent 24 days in the house before being tested and was asymptomatic. Physical overcrowding in the house provided a favourable environment for intra-cluster infection transmission. Restriction of movement of family members due to countrywide lockdown limited the spread in community. Among the infected, only four individuals developed symptoms. The complete genome sequences of SARS-CoV-2 was retrieved using next-generation sequencing from eight clinical samples which demonstrated a 99.99% similarity with reference to Wuhan strain and the phylogenetic analysis demonstrated a distinct cluster, lying in the B.6.6 pangolin lineage.

Microbiome, the study of microbial communities in specific environments, has developed significantly since the Human Microbiome Project began. Microbiomes have been associated with changes within environmental niches and the development of various diseases. The development of high-throughput technology such as next-generation sequencing has also allowed us to perform transcriptome studies, which provide accurate functional profiling data. Metabolome studies, which analyse the metabolites found in the environment, are the most direct environmental condition indicator. Although each dataset provides valuable information on its own, the integration of multiple datasets provides a deeper understanding of the relationship between the host, agent and environment. Therefore, network analysis using multiple datasets might give a clearer understanding of disease pathogenesis.

The finding of conjoined oocytes is a rare occurrence that accounts for only 0.3% of all human retrieved oocytes. This phenomenon is quite different from that of a traditional single oocyte emanating from one follicle, and may result in dizygotic twins and mosaicism. Given the insufficient evidence on how to approach conjoined oocytes, their fate is variable among different in vitro fertilization (IVF) centres. In this observational report, we propose a new protocol for the use of these conjoined oocytes using intracytoplasmic sperm injection (ICSI), laser-cutting technique and next-generation sequencing (NGS). The first case report demonstrates that conjoined oocytes can penetrate their shared zona pellucida (ZP) at Day 6. The second case is that of a 25-year-old female patient who underwent a successful embryo transfer cycle after removal of one oocyte in which a pair of conjoined human oocytes underwent ICSI, laser-cutting separation and NGS testing. The patient achieved pregnancy and gave birth to single healthy female originally derived from conjoined oocytes. This case provided a means through which normal pregnancy may be achieved from conjoined oocytes using laser-cutting separation techniques. The protocol described may be especially beneficial to patients with a limited number of oocytes.

The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Paediatric cardiomyopathy is a progressive and often lethal disorder and the most common cause of heart failure in children. Despite their severe outcomes, their genetic etiology is still poorly characterised. The current study aimed at uncovering the genetic background of idiopathic primary hypertrophic cardiomyopathy in a cohort of Egyptian children using targeted next-generation sequencing. The study included 24 patients (15 males and 9 females) presented to the cardiomyopathy clinic of Cairo University Children’s Hospital with a median age of 2.75 (0.5–14) years. Consanguinity was positive in 62.5% of patients. A family history of hypertrophic cardiomyopathy was present in 20.8% of patients. Ten rare variants were detected in eight patients; two pathogenic variants (8.3%) in MBPC3 and MYH7, and eight variants of uncertain significance in MYBPC3, TTN, VCL, MYL2, CSRP3, and RBM20.

Here, we report on the first national study in Egypt that analysed sarcomeric and non-sarcomeric variants in a cohort of idiopathic paediatric hypertrophic cardiomyopathy patients using next-generation sequencing. The current pilot study suggests that paediatric hypertrophic cardiomyopathy in Egypt might have a particular genetic background, especially with the high burden of consanguinity. Including the genetic testing in the routine diagnostic service is important for a better understanding of the pathophysiology of the disease, proper patient management, and at-risk detection. Genome-wide tests (whole exome/genome sequencing) might be better than the targeted sequencing approach to test primary hypertrophic cardiomyopathy patients in addition to its ability for the identification of novel genetic causes.

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin–Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually “nonfilamentous” virus strains.

Southern crabgrass [Digitaria ciliaris (Retz.) Koeler] is an annual grass weed that commonly infests turfgrass, roadsides, wastelands, and cropping systems throughout the southeastern United States. Two biotypes of D. ciliaris (R1 and R2) with known resistance to cyclohexanediones (DIMs) and aryloxyphenoxypropionates (FOPs) previously collected from sod production fields in Georgia were compared with a separate susceptible biotype (S) collected from Alabama for the responses to pinoxaden and to explore the possible mechanisms of resistance. Increasing rates of pinoxaden (0.1 to 23.5 kg ha−1) were evaluated for control of R1, R2, and S. The resistant biotypes, R1 and R2, were resistant to pinoxaden relative to S. The S biotype was completely controlled at rates of 11.8 and 23.5 kg ha−1, resulting in no aboveground biomass at 14 d after treatment. Pinoxaden rates at which tiller length and aboveground biomass would be reduced 50% (I50) and 90% (I90) for R1, R2, and S ranged from 7.2 to 13.2 kg ha−1, 6.9 to 8.6 kg ha−1, and 0.7 to 2.1 kg ha−1, respectively, for tiller length, and 7.7 to 10.2 kg ha−1, 7.2 to 7.9 kg ha−1, and 1.6 to 2.3 kg ha−1, respectively, for aboveground biomass. Prior selection pressure from DIM and FOP herbicides could result in the evolution of D. ciliaris cross-resistance to pinoxaden herbicides. Amplification of the carboxyl-transferase domain of the plastidic ACCase by standard PCR identified a point mutation resulting in an Ile-1781-Leu amino acid substitution only for the resistant biotype, R1. Further cloning of PCR product surrounding the 1781 region yielded two distinct ACCase gene sequences, Ile-1781 and Leu-1781. The amino acid substitution, Ile-1781-Leu in both resistant biotypes (R1 and R2), however, was revealed by next-generation sequencing of RNA using Illumina platform. A point mutation in the Ile-1781 codon leading to herbicide insensitivity in the ACCase enzyme has been previously reported in other grass species. Our research confirms that the Ile-1781-Leu substitution is present in pinoxaden-resistant D. ciliaris.

Tick-borne diseases caused by Theileria are of economic importance in domestic and wildlife ruminants. The majority of Theileria infects a limited number of host species, supporting the concept of host specificity. However, some Theileria seem to be generalists challenging the host specificity paradigm, such as Theileria sp. (sable) reported from various vertebrate hosts, including African buffalo, cattle, dogs and different antelope species. We tested the hypothesis that T. sp. (sable) uses Bovidae as hosts in general using a real-time polymerase chain reaction assay specific for T. sp. (sable) and a closely related genotype: T. sp. (sable-like). Various antelope species from the Tragelaphini (black wildebeest, blesbuck, blue wildebeest, gemsbuck, sable and waterbuck) tested positive for either T. sp. (sable) or T. sp. (sable-like). However, no African buffalo (n = 238) or cattle (n = 428) sampled in the current study tested positive, suggesting that these latter species are not carrier hosts. The results were confirmed using next-generation sequencing which also indicated at least 13 new genotypes or species found in various antelope and giraffes. Genotypes were found in single host species or in evolutionarily related hosts, suggesting that host specificity in Theileria may be a lineage specific phenomenon likely associated with tick-host-parasite co-evolution.