Genome Sequence Analysis Research Articles

A retrospective whole genome sequencing of SARS-CoV-2 isolate from respiratory sample of an individual from Ota – Ogun State, Nigeria

Severe acute respiratory syndrome coronavirus – 2 (SARS-CoV-2) which was responsible for the COVID-19 pandemic has now been considered an endemic virus, that will continually produce sporadic outbreaks in different communities around the globe. Although, there are several surveillance studies on SARS-CoV-2 globally, including Nigeria, there is still an important need to understand the uniqueness of the strains of the virus that was circulating immediately after the pandemic, to add to the global database of information that will aid vaccine production and future preparedness against another SARS - related CoV pandemic.Towards the end of the pandemic, between February – August 2022, SARS-CoV-2 was detected in a surveillance project in Ota – Ogun State Nigeria. We carried out a retrospective whole genome sequencing (WGS) analysis of SARS-CoV-2 in the previously reported positive sample, at Inqaba biotech in South Africa. RNA extraction was carried out using the Quick – RNA viral kit (Zymo) and library preparation was done by NEBNext ARTIC SARS-CoV-2 FS Library Prep kit (Illumina) according to the manufacturer's instruction. The WGS was carried out on the NextSeq500 platform by Illumina. The fastq file containing the unassembled raw sequence reads were submitted to NCBI SARS-CoV-2 resources, and a BioProject accession number PRJNA1076330 was issued. The DRAGEN Targeted Microbial, GISAID-CoVsurver mutation, BLAST and MAFFT applications were used for analysis.The spike (s) gene of the study sequence possessed seven mutations (G142D, A163V, V213G, D614G, H655Y, N679K, P681H) and shared 99.4 % identity with those of the Wuhan reference sequence WIV04. The non-structural proteins (NSP) – 7,8,9,10 and 16 shared 100 % identity with bat/Yunnan/RaTG13 (a SARS- related CoV found in bats) sequence on GISAID. Although, the study sequence was obtained from an asymptomatic individual, the S mutations observed are known to be related to virulence, antigenic shift, enhanced transmissibility and host change. Thus, upon successful exploitation of this asymptomatic variant, it may possibly be transformed to a potential candidate for SARS-CoV-2 vaccine in future.

Occurrence of Uncultured Legionella spp. in Treated Wastewater Effluent and Its Impact on Human Health (SCA.Re.S Project).

Wastewater treatment plants (WWTPs) provide optimal conditions for the environmental spread of Legionella. As part of the Evaluation of Sanitary Risk Related to the Discharge of Wastewater to the Ground (SCA.Re.S) project, this study was conducted to evaluate the presence of Legionella in WWTP effluent and in groundwater samples collected from two wells located downstream from the plant. The samples were analyzed to determine the concentrations of Legionella spp using the standard culture-based method and molecular techniques, followed by genomic sequencing analysis. Legionella was detected only with the molecular methods (except in one sample of effluent positive for L. pneumophila serogroup 6), which showed viable Legionella pneumophila and L. non-pneumophila through the use of free DNA removal solution in both the effluent and groundwater, with concentrations that progressively decreased downstream from the plant. Viable L. pneumophila appeared to be slightly more concentrated in warm months. However, no significant differences (p ≥ 0.05) in concentrations between cold and warm months were observed. A genotypic analysis characterized the species present in the samples and found that uncultured Legionella spp, as yet undefined, constituted the prevalent species in all the samples (range 77.15-83.17%). WWTPs play an important role in the hygienic and sanitary quality of groundwater for different uses. The application of Legionella control systems during the purification of effluents is warranted to prevent possible outbreaks of legionellosis.

STUDY OF SOMATIC AND GERMINAL MUTATIONS IN ARTERIOVENOUS MALFORMATIONS OF THE BRAIN USING WHOLE EXOME SEQUENCING

Rare vascular lesions known as brain arteriovenous malformations (bAVMs) are caused by shunts between cerebral arteries and veins that do not involve the capillary bed. The majority of bAVMs do not cause any symptoms, however some can be controlled by seizures and result in potentially fatal brain hemorrhages. Over the past ten years, significant advancements in our understanding of bAVM pathogenesis have been accomplished, primarily through the use of genome sequencing and biomolecular analysis. An autosomal dominant vascular disorder called Hereditary Hemorrhagic Telangiectasia (HHT, also known as Rendu-Osler-Weber Syndrome) affects about 1 in 5000 people globally and is associated with about 5% of brain AVMs, while the remaining 95% are due to sporadic mutations. The major aim of the project is to investigate genetic causes of sporadic forms of AVMs in the brain by means of whole-exome sequencing of tissues with AVM lesions and blood samples. Analysis of somatic mutations will broaden the understanding of AVM pathogenesis. This study was carried out in accordance with the Declaration of Helsinki's tenets and approved by the National Center of Biotechnology 's ethics committee (№9, 7 November 2023, ) Astana). Based on clinical data, the age range of the patients at the onset of the disease also encompassed 25 to 40 years. To confirm the diagnosis, MRI and CAG data were used to validate the diagnosis. Each patient completed a specially designed questionnaire with medical information (such as age of debut and type of course) and demographic data (such as age, gender, and nationality). Three patients were classified as having a Spetzler-Martin grade II, while one patient was categorized as grade III. The sizes of arteriovenous malformations (AVMs) varied from 0 to 3 mm in one patient (grade II) to 3 to 6 mm in three patients (grades II-III). AVM tissue specimens were obtained during clinically necessary procedures. Brain AVM tissues were collected during surgery, which involved trepanation and excision of the AVM. Blood samples were also collected from these patients. Both DNA and RNA were isolated from these tissue and blood samples for subsequent exome sequencing an. Qubit 2.0 fluorometer was employed for more precise determination of DNA concentration, given that the nucleic acids will be utilized in future whole exome sequencing analysis. Whole exome sequencing was performed on six samples (blood and tissue) by Novaseq 6000 (Illumina). The annotated results were analyzed for the presence of single-nucleotide polymorphisms (SNP) and insertion-deletion mutations (Indels) between DNAs from the AVM tissue sample and blood. The GATK workflow was used for the analysis and annotation of the raw data, and vcf files were generated. Then, the variants were filtered for a quality ≥ 30 in Excel 1st blood 117554, 1st tissue 114715; 2nd blood 112972, 2nd tissue 116022; 3rd blood 112859, 3rd tissue 117168 and genotype differences between tissue and blood DNA samples 1st blood 114512, 1st tissue 111682; 2nd blood 110614, 2nd tissue 111982; 3rd blood 110564, 3rd tissue 112805. After selecting only non-synonymous, frameshift insertions and deletions variants with different genotypes in the blood and tissue samples, 1st blood 279, 1st tissue 277; 2nd blood 279, 2nd tissue 271; 3rd blood 275, 3rd tissue 268 variations were observed. Analysis of whole-exome data continues.

Distribution rules of 8-mer spectra and characterization of evolution state in animal genome sequences

BackgroundStudying the composition rules and evolution mechanisms of genome sequences are core issues in the post-genomic era, and k-mer spectrum analysis of genome sequences is an effective means to solve this problem.ResultWe divided total 8-mers of genome sequences into 16 kinds of XY-type due to XY dinucleotides number in 8-mers. Previous works explored that the independent unimodal distributions observed only in three CG-type 8-mer spectra, while non-CG type 8-mer spectra have not the universal phenomenon from prokaryotes to eukaryotes. On this basis, we analyzed the distribution variation of non-CG type 8-mer spectra across 889 animal genome sequences. Following the evolutionary order of animals from primitive to more complex, we found that the spectrum distributions gradually transition from unimodal to tri-modal. The relative distance from the average frequency of each non-CG type 8-mers to the center frequency is different within a species and among different species. For the 8-mers contain CG dinucleotides, we further divided these into 16 subsets, where each 8-mer contains both CG and XY dinucleotides, called XY1_CG1 subsets. We found that the separability values of XY1_CG1 spectra are closely related to the evolution and specificity of animals. Considering the constraint of Chargaff’s second parity rule, we finally obtained 10 separability values as the feature set to characterize the evolution state of genome sequences. In order to verify the rationality of the feature set, we used 14 common classification algorithms to perform binary classification tests. The results showed that the accuracy (Acc) ranged between 98.70% and 83.88% among birds, other vertebrates and mammals.ConclusionWe proposed a credible feature set to characterizes the evolution state of genomes and obtained satisfied results by the feature set on large scale classification of animals.

Genomic Characterization of Phage ZP3 and Its Endolysin LysZP with Antimicrobial Potential against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3's endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal-arrest-release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage-Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.

Whole genome sequence analysis of turkey orthoreovirus isolates reveals a strong viral host-specificity and naturally occurring co-infections in commercial turkeys

Avian orthoreoviruses (ARV) are an emerging threat for the poultry industry, both in the United States (US) and globally. ARV infections in turkeys have been associated with arthritis, lameness and neurological disorders, and cost the US economy approximately USD 33 million per year. There is not a commercial vaccine available and the shortage of turkey ARV (TRV) genomic data hinders the efforts to explore the molecular epidemiology of this virus, although several studies suggest a close relationship between European TRVs and TRVs present in the US. This study shows a snapshot of the genomic diversity of Avian orthoreoviruses (ARV) circulating in Germany in the mid-2000s. Through a deep genomic analysis of 18 ARV isolates recovered from sick turkeys, we observed that co-infection was a common condition. 80% of the samples showed signs of a simultaneous infection with a TRV and a chicken ARV (CRV). We believe this is the first reported evidence of CRV and TRV naturally occurring co-infections in commercial turkeys. These co-infection events were identified due to the significant genomic diversity observed among ARV infecting various production bird species. Our phylogenetic analysis revealed a consistent host-associated ARV clustering, with three main clades: (i) a TRV clade, (ii) a CRV clade, and (iii) a Duck ARV (DRV)/Goose ARV (GRV) clade. Furthermore, our findings indicate that German TRVs have interacted with their European and American counterparts, suggesting active mobilization of the virus, likely through the commercial trading of live animals. However, we also consider the potential role of migratory birds in the international movement of ARV.

Pathogenic and genomic characterization of Ostreid herpesvirus 1 associated with mass mortalities of blood cockle (Anadara kagoshimensis)

Ostreid herpesvirus 1 (OsHV-1) is an infectious viral disease of bivalve mollusks with a wide geographic distribution across five continents. Since its initial identification in 1991, the disease has been identified as the cause of mortality in more than ten species of bivalve mollusks. The transmission of OsHV-1 is thought to be facilitated by water. In 2019 and 2021, mass mortalities of blood cockle (Anadara kagoshimensis) hatchery broodstock associated with OsHV-1 infection were reported. The presence of OsHV-1 DNA was confirmed in all hatchery samples with levels exceeding 106 copies per milligram of DNA. OsHV-1 was not detected in the wild populations from which the broodstock was collected, indicating that the virus was introduced into the hatchery with seawater from the open ocean. The results of the histopathological examination indicated that tissue lesions and associated hemocyte infiltration were most prevalent in the connective tissues of the digestive tract, mantle, gills and foot of the diseased A. kagoshimensis. In situ hybridization (ISH) signals of OsHV-1 DNA were frequently observed in infiltrated hemocytes, putative fibroblasts, and on occasion, in muscle cells with weak staining. Transmission electron microscopy analysis demonstrated the presence of herpes-like viral particles within the nucleus of infected cells. The genomes of the OsHV-1 variants from cases 2019 and 2021 were successfully resolved through direct high-throughput sequencing of infected tissues. A phylogenetic analysis of the complete genome sequences of 32 OsHV-1 variants revealed that the segregation of the virus was primarily structured by host species and geographic origin, with some exceptions. We propose that the sympatric distribution of different host species and the potential occurrence of cross-species transmission of OsHV-1 are responsible for the inconsistency between the phylogenetic segregation and their host species. This is the first report on the pathogenic and genomic characterization of OsHV-1 infecting A. kagoshimensis. The epidemiological and phylogenetic analyses offer new insights into the evolution of the virus and provide valuable information for the prevention and control of the disease.

Comparison of Mycobacterium tuberculosis Strains with H37Rv Using Whole Genome Sequence Analysis to Identify Virulence Factors and Phylogenetic Relationships

Mycobacterium tuberculosis (Mtb) poses a significant disease burden in developing countries, largely due to the rising issue of drug resistance. In Pakistan, the genetic and molecular characteristics of resistant strains have not been thoroughly analyzed or compared with the reference Mtb strain, H37Rv, to understand the molecular and genetic epidemiology. Mtb employs various strategies to resist anti-tuberculosis drugs, and antimicrobial resistance, including single and multidrug resistance, has increased extensively in developing nations like Pakistan. In this study, the whole genomes of three Mtb strains (335, 335-2, and 311) were uploaded to the Rapid Annotation Subsystems Technology (RAST) server to retrieve coding DNA sequences (CDS). The RAST server identified 4,454, 4,445, and 4,396 CDS for Mtb strains 335, 335-2, and 311, respectively. Mutation analysis was performed using NCBI nucleotide BLAST and CLUSTAL Omega, comparing the sequences with the H37Rv reference strain. The analysis revealed 909, 832, and 1,135 mutations in the 335, 335-2, and 311 strains, respectively. Phylogenetic analysis based on the most virulent protein (EccC3) indicated a close resemblance between all three strains and the H37Rv strain. Furthermore, Mycobacteriumcanettii (accession number WP_203037448.1) was identified as the closest related bacterium to the studied strains and H37Rv.

Porcine-derived pancreatic enzyme replacement therapy may be linked to chronic hepatitis E virus infection in cystic fibrosis lung transplant recipients

ObjectivesIn high-income countries hepatitis E virus (HEV) is an uncommonly diagnosed porcine-derived zoonoses. After identifying disproportionate chronic HEV infections in persons with cystic fibrosis (pwCF) postlung transplant, we sought to...

Complete genome sequence analysis of Pestalotiopsis microspora, a fungal pathogen causing kiwifruit postharvest rots

BackgroundThe postharvest rot of kiwifruit is one of the most devastating diseases affecting kiwifruit quality worldwide. However, the genomic basis and pathogenicity mechanisms of kiwifruit rot pathogens are lacking. Here we report the first whole genome sequence of Pestalotiopsis microspora, one of the main pathogens causing postharvest kiwifruit rot in China. The genome of strain KFRD-2 was sequenced, de novo assembled, and analyzed.ResultsThe genome of KFRD-2 was estimated to be approximately 50.31 Mb in size, with an overall GC content of 50.25%. Among 14,711 predicted genes, 14,423 (98.04%) exhibited significant matches to genes in the NCBI nr database. A phylogenetic analysis of 26 known pathogenic fungi, including P. microspora KFRD-2, based on conserved orthologous genes, revealed that KFRD-2’s closest evolutionary relationships were to Neopestalotiopsis spp. Among KFRD-2’s coding genes, 870 putative CAZy genes spanned six classes of CAZys, which play roles in degrading plant cell walls. Out of the 25 other plant pathogenic fungi, P. microspora possessed a greater number of CAZy genes than 22 and was especially enriched in GH and AA genes. A total of 845 transcription factors and 86 secondary metabolism gene clusters were predicted, representing various types. Furthermore, 28 effectors and 109 virulence-enhanced factors were identified using the PHI (pathogen host-interacting) database.ConclusionThis complete genome sequence analysis of the kiwifruit postharvest rot pathogen P. microspora enriches our understanding its disease pathogenesis and virulence. This study establishes a theoretical foundation for future investigations into the pathogenic mechanisms of P. microspora and the development of enhanced strategies for the efficient management of kiwifruit postharvest rots.

Molecular epidemiology of brucellosis in Asia: insights from genotyping analyses.

Brucellosis infects humans and animals worldwide but is particularly prevalent in Asia. In many Asian countries, molecular diagnostic tools for accurate molecular diagnostics and molecular epidemiology are lacking. Nonetheless, some countries have conducted in-depth molecular epidemiological studies. The objective of this study was to reveal the genetic relationships, geographic origins, and distributions of Brucella strains across Asia for two primary species, B. abortus and B. melitensis. For this, we systematically searched genotyping data from published studies on the molecular epidemiology of Brucella species for both humans and livestock in Asia. We used data from multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing analysis of Brucella strains. We also analyzed the MLVA genotypes of 129 B. abortus isolates and 242 B. melitensis isolates with known origins in Asia from an online MLVA database using MLVA-11 data in minimum spanning trees and MLVA-16 data in neighbor-joining trees. We found that the B. melitensis East Mediterranean lineage is predominant across the continent, with only a small number of samples from the Africa and Americas lineages, and none from the West Mediterranean lineage. The "abortus C" genotype was the most common group of B. abortus in Asia, with limited genetic variation for this species. Several studies also reported that Near Eastern countries frequently encounter human brucellosis cases of B. abortus from genotypes 42 and 43. Our study highlights the inconsistent collection of genetic data for Brucella species across Asia and a need for more extensive sampling in most countries. Finally, a consistent nomenclature is necessary to define various groupings of strains within a lineage (i.e., clade) so uniform terminology should denote particular genetic groups that are understood by all researchers.

New Viruses Infecting Hyperthermophilic Bacterium Thermus thermophilus.

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.

Whole genome sequence analysis reveals high genomic diversity and potential host-driven adaptations among multidrug-resistant Escherichia coli from pre-weaned dairy calves.

Food-producing animals such as dairy cattle are potential reservoirs of antimicrobial resistance (AMR), with multidrug-resistant (MDR) organisms such as Escherichia coli observed in higher frequency in young calves compared to older cattle. In this study, we characterized the genomes of enteric MDR E. coli from pre-weaned dairy calves with and without diarrhea and evaluated the influence of host-level factors on genomic composition. Whole genome sequence comparative analysis of E. coli (n = 43) revealed substantial genomic diversity that primarily clustered by sequence type and was minimally driven by calf diarrheal disease status (healthy, diarrheic, or recovered), antimicrobial exposure, and dietary zinc supplementation. Diverse AMR genes (ARGs)-including extended-spectrum beta-lactamase genes and quinolone resistance determinants-were identified (n = 40), with unique sets of ARGs co-occurring in gene clusters with large AMR plasmids IncA/C2 and IncFIB(AP001918). Zinc supplementation was not significantly associated with the selection of individual ARGs in E. coli, however analysis of ARG and metal resistance gene pairs identified positive associations between certain aminoglycoside, beta-lactam, sulfonamide, and trimethoprim ARGs with acid, tellurium and mercury resistance genes. Although E. coli in this study lacked the typical virulence factors of diarrheagenic strains, virulence genes overlapping with those in major pathotypes were identified. Among the 103 virulence genes detected, the highest abundance and diversity of genes corresponded to iron acquisition (siderophores and heme uptake). Our findings indicate that the host-level factors evaluated in this study were not key drivers of genomic variability, but that certain accessory genes in enteric MDR E. coli may be enriched. Collectively, this work provides insight into the genomic diversity and host-microbe interface of MDR E. coli from pre-weaned dairy calves.

Molecular and biochemical characterization of a plant-like iota-class glutathione S-transferase from the halotolerant cyanobacterium Halothece sp. PCC7418.

This study identifies a unique glutathione S-transferase (GST) in extremophile using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis. Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress. Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses.

Genome variations in sea cucumbers: Insights from genome survey sequencing and comparative analysis of mitochondrial genomes

Sea cucumbers, marine benthic invertebrates, play crucial roles in maintaining the stability of marine ecosystems and hold key evolutionary positions. However, information regarding their genomes remains limited. Here, we conducted genome survey analyses on seven species from four orders. Results indicated that Colochirus anceps, Colochirus quadrangularis, and Pseudocolochirus violaceus within the order Dendrochirotida have significantly larger genomes (2238–3754 Mbp) compared to conventional sea cucumber genomes, accompanied by a very high proportion of repeat sequences (69.39–72.52 %). While Holothuria edulis and Holothuria atra exhibited similar genome sizes comparable to those of other species within the order Holothuriida, heterozygosity and repeat content varied among all the six species in this order. The representative species Apostichopus californicus of the order Synallactida possesses the smallest genome size (573.45Mbp) within its order, but its heterozygosity (2.24 %) is significantly higher than that of other species. The representative species Synapta maculata of the order Apodida exhibited a normal genome size (900.97 Mbp), lower proportion of repeat sequences (42.19 %), and lower heterozygosity (0.84 %), making it the species with the least challenges for genome sequencing and assembly in the future among all surveyed species. Subsequently, we compiled genomic information from a total of 19 sea cucumber genomes, both newly sequenced and previously reported, revealing a significant linear relationship (P = 0.0001) between genome size and the proportion of repeat sequences in sea cucumbers. Additionally, phylogenetic and comparative analysis of mitochondrial genomes among them indicated extensive rearrangements within the order Apodida, leading to significant discrepancies between mitochondrial and nuclear genome phylogenies.

Molecular characterization of avian metapneumovirus subtype C associated with hydrosalpinx fluid and egg drop syndrome in Jinding ducks.

A disease called "hydrosalpinx fluid and egg drop syndrome" (HFEDS) was observed in four flocks of Jinding ducks (Anas platyrhynchos domesticus) in Northeast China during the years 2022 to 2023. Here, we investigated the possible involvement of avian metapneumovirus (AMPV) infection. Full-length genome sequencing and sequence analysis of two AMPV strains showed that they belong to Eurasian lineage of AMPV subtype C. Based on surface glycoprotein (G) sequence comparisons, the Eurasian lineage can be divided into two sublineages (E1 and E2), and sublineage E2 is circulating in Jinding duck populations in Northeast China.

Genome Sequencing and Phylogenetic Analysis of SARS-CoV-2 Isolated from Patients with COVID-19 in Hospital Canselor Tuanku Muhriz (HCTM), Malaysia

Genome Sequencing and Phylogenetic Analysis of SARS-CoV-2 Isolated from Patients with COVID-19 in Hospital Canselor Tuanku Muhriz (HCTM), Malaysia

Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae.

Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of Enterobacter asburiae CGJ001 with high hyaluronan lyase activity was screened from industrial wastewater. This strain exhibited an impressive enzyme activity of 40,576 U/mL after being incubated for 14 h. Whole genome sequencing analysis revealed that E. asburiae CGJ001 contained a cluster of genes involved in HA degradation, transport, and metabolism. A newly identified enzyme responsible for glycosaminoglycan degradation was designated as HylEP0006. A strain of E. coli BL21(DE3)/pET-22b(+)-hylEP0006 was successfully constructed. HylEP0006 exhibited optimal degradation at 40 °C and pH 7.0, showing a high activity of 950,168.3 U/mg. HylEP0006 showed specific activity against HA. The minimum degradation fragment of HylEP0006 was hyaluronan tetrasaccharides, and HylEP0006 could efficiently degrade HA into unsaturated disaccharides (HA2), with HA2 as the final product. These characteristics indicate that HylEP0006 has a potential application prospect for the extraction and utilization of hyaluronic acid.

Dimensionality reduction distills complex evolutionary relationships in seasonal influenza and SARS-CoV-2.

Public health researchers and practitioners commonly infer phylogenies from viral genome sequences to understand transmission dynamics and identify clusters of genetically-related samples. However, viruses that reassort or recombine violate phylogenetic assumptions and require more sophisticated methods. Even when phylogenies are appropriate, they can be unnecessary or difficult to interpret without specialty knowledge. For example, pairwise distances between sequences can be enough to identify clusters of related samples or assign new samples to existing phylogenetic clusters. In this work, we tested whether dimensionality reduction methods could capture known genetic groups within two human pathogenic viruses that cause substantial human morbidity and mortality and frequently reassort or recombine, respectively: seasonal influenza A/H3N2 and SARS-CoV-2. We applied principal component analysis (PCA), multidimensional scaling (MDS), t-distributed stochastic neighbor embedding (t-SNE), and uniform manifold approximation and projection (UMAP) to sequences with well-defined phylogenetic clades and either reassortment (H3N2) or recombination (SARS-CoV-2). For each low-dimensional embedding of sequences, we calculated the correlation between pairwise genetic and Euclidean distances in the embedding and applied a hierarchical clustering method to identify clusters in the embedding. We measured the accuracy of clusters compared to previously defined phylogenetic clades, reassortment clusters, or recombinant lineages. We found that MDS embeddings accurately represented pairwise genetic distances including the intermediate placement of recombinant SARS-CoV-2 lineages between parental lineages. Clusters from t-SNE embeddings accurately recapitulated known phylogenetic clades, H3N2 reassortment groups, and SARS-CoV-2 recombinant lineages. We show that simple statistical methods without a biological model can accurately represent known genetic relationships for relevant human pathogenic viruses. Our open source implementation of these methods for analysis of viral genome sequences can be easily applied when phylogenetic methods are either unnecessary or inappropriate.

Molecular characteristic, evolution, and pathogenicity analysis of avian infectious bronchitis virus isolates associated with QX type in China

Infectious bronchitis virus (IBV) is one of the major avian pathogens plaguing the global poultry industry. Although vaccination is the primary preventive measure for IBV infection, the emergence of virus variants with mutations and recombination has resulted in IBV circulating globally, presenting a challenge for IB control. Here, we isolated 3 IBV strains (CZ200515, CZ210840, and CZ211063) from suspected sick chickens vaccinated with IBV live attenuated vaccines (H120, 4/91, or QXL87). Phylogenetic analysis of the S1 gene sequence of the spike (S) revealed that the 3 isolates belonged to the QX-type (GI-19 lineage). Whole genome sequencing and recombination analysis indicated that CZ200515 and CZ210840 contained genetic material from 4/91 and Scyz3 (QX-type), possibly due to recombination between the circulating strain and the 4/91 vaccine strain, while no evidence of recombination was found in CZ211063. Pathogenicity analysis in 1-day-old specific pathogen-free (SPF) chickens demonstrated that all 3 isolates caused severe tissue damage and varying degrees of mortality. Virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and the QX-type vaccine strain (QXL87). Amino acid sequence homology analysis of S1 revealed 5%-6.5% variances between the isolates and QXL87. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the hypervariable region (HVR) and the neutralizing epitope region resulted in antigenic variation in isolates by changing the antigen conformation. Our data indicate antigenicity variances between QX isolates and QXL87 vaccine strains, potentially resulting in immune evasion occurrences. Overall, these results offer crucial insights into the epidemiology and pathogenicity of QX-type IBV, facilitating improved selection and formulation of vaccines for disease management.