Complete Coding Region Sequences Research Articles

Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing.

Rapid and reliable detection tools are essential for disease surveillance and outbreak management, and genomic data is essential to determining pathogen origin and monitoring of transmission pathways. Low virus copy number and poor RNA quality can present challenges for genomic sequencing of plant viruses, but this can be overcome by enrichment of target nucleic acid. A targeted whole genome sequencing (TWG-Seq) approach for the detection of cucumber green mottle mosaic virus (CGMMV) has been developed where overlapping amplicons generated using two multiplex RT-PCR assays are then sequenced using the Oxford Nanopore MinION. Near complete coding region sequences were assembled with ≥100× coverage for infected leaf tissue dilution samples with RT-qPCR cycle quantification (Cq) values from 11.8 to 38 and in seed dilution samples with Cq values 13.8 to 27. Consensus sequences assembled using this approach showed greater than 99% nucleotide similarity when compared to genomes produced using metagenomic sequencing. CGMMV could be confidently detected in historical seed isolates with degraded RNA. Whilst limited access to, and costs associated with second-generation sequencing platforms can influence diagnostic outputs, the portable Nanopore technology offers an affordable high throughput sequencing alternative when combined with TWG-Seq for low copy or degraded samples.

The first evidence of shaking mink syndrome-astrovirus associated encephalitis in farmed minks, China.

A novel neurological disorder, shaking mink syndrome (SMS), emerged in Denmark and Sweden in 2000. SMS has seldom been reported in China, but the causative agent has not been detected in the country. SMS outbreaks occurred in multiple provinces in 2020. A total of 44 brain samples from minks associated with SMS were collected from Heilongjiang, Liaoning and Shandong provinces of which 28 samples (63.3%) were SMS-astrovirus (SMS-AstV)-positive by reverse transcription PCR. Histopathological examination revealed non-suppurative encephalitis in three minks. Moreover, the complete coding region sequences (CDSs, 6559 bp) of a sample collected from a 2-month-old mink (termed SMS-AstV-H1, GSA accession No. SAMC816786) were amplified by PCR and Sanger sequencing. The complete CDS and open reading frame 2 sequences of SMS-AstV-H1 were 94.3% and 96.4% identical to an SMS-AstV strain (GenBank accession number: GU985458). Phylogenetically, SMS-AstV-H1 was closely related to an SMS-AstV strain (GU985458). Based on the above results, we describe SMS-AstV-associated encephalitis in farmed minks in China. Future studies need to focus on epidemiology, virus isolation and potential interspecies transmission of SMS-AstV.

Possible role of the CYC2c gene in the cornflower-like ray floret phenotype of Gaillardia cultivars.

Gaillardia plants have been widely cultivated in China and have become an important component of garden landscaping. Different from the common ligulate ray floret, the cornflower-like (funnel-shaped) ray floret is a special phenotype variation in Gaillardia species. Previous studies revealed that CYC-like genes could shape the floret phenotype in Compositae. To reveal the molecular mechanism of the cornflower-like phenotype, we checked the capitulum transcriptomes of several cultivars of Gaillardia that possess different ray florets. As a result, we identified 11 CYC-like genes, of which five included complete coding region sequences. Phylogenetic analysis revealed that all five genes were CYC2-like genes. Relative expression analysis of RNA-resequencing reads, qRT-PCR comparison, and gene-silencing treatment all showed that the CYC2c gene is the main genetic mechanism responsible for the shaping of the cornflower-like ray floret phenotype in Gaillardia cultivars. This study expounded our understanding of flower morphology evolution and provides useful insights for improving Compositae breeding.

Detection of Systemic Canine Kobuvirus Infection in Peripheral Tissues and the Central Nervous System of a Fox Infected with Canine Distemper Virus.

Canine kobuvirus (CaKV) is a globally distributed pathogen of dogs and is predominantly associated with infection of the gastrointestinal tract. However, an etiological link to enteric disease has not been established since CaKV has been identified in both asymptomatic dogs and animals with diarrheic symptoms. In this study, an extraintestinal CaKV infection was detected by next-generation sequencing in a fox (Vulpes vulpes) in Germany concomitant with a canine distemper virus (canine morbillivirus; CDV) co-infection. Phylogenetic analysis of the complete coding region sequence showed that this strain was most closely related to a CaKV strain detected in a dog in the United Kingdom in 2008. The tissue and cellular tropism of CaKV was characterized by the detection of viral antigens and RNA. CaKV RNA was detected by in situ hybridization in different tissues, including epithelial cells of the stomach and ependymal cells in the brain. The use of a new RT-qPCR assay for CaKV confirmed the systemic distribution of CaKV with viral RNA also detected in the lymph nodes, bladder, trachea, and brain. The detection of a CDV infection in this fox suggests that immunosuppression should be further investigated as a contributing factor to the enhanced extraintestinal spread of CaKV.

Complete genome sequencing and evolutionary analysis of HCV subtype 6xg from IDUs in Yunnan, China.

BackgroundHCV genotype 6 (HCV-6) typically circulates in Southeast Asia and exhibits the highest genetic diversity among the eight HCV genotypes. In our previous work, a group of HCV-6 sequences was not clearly classified. Here, we further characterized this HCV-6 variant and analyzed the evolutionary history of the enlarged HCV-6 family.MethodsBlood samples from eight HCV seropositive samples collected from intravenous drug users (IDUs) in 2014 in Yunnan Province, China. The full-length HCV genome sequences were amplified by using reverse transcription PCR followed by DNA sequencing and phylogenetic analysis. Bayesian evolutionary analysis was performed with the complete coding region sequences of subtype 6a-6xh.ResultsThe eight genomes had the same coding region of 9051 nucleotides. The complete coding region sequences of the eight HCV isolates formed a distinct phylogenetic group from the previously assigned HCV-6 subtypes (6a-6xf), however which clustered with 6xg reference sequences that were found in Kachin State, Myanmar, and recently assigned and released. The p-distances of the eight isolates to subtype 6a-6xf and 6xh ranged from 0.143 to 0.283. Based on the HCV-6 complete coding region sequences, we constructed a timescaled phylogenetic tree to reveal the HCV-6 evolutionary history, in which there were four HCV-6 phylogenetic subsets, whose median tMRCAs were 294.8, 388.5, 348.5 and 197.0 years ago, respectively. Subtype 6xg clustered into Subset I, and had the most recent common ancestor with subtype 6n, which dated back to 101.2 (95% HPD: 78.7, 125.8) years ago. The genetic evolutionary analysis further confirmed that subtype 6xg originated from Myanmar, and transmitted to Dehong through cross-border IDUs.ConclusionThe HCV-6 variant characterized in this study belonged to newly assigned subtype 6xg. Our finding further confirmed the assignment of 6xg. HCV-6 family was highly divers and had a complicated evolutionary history in Southeast Asia. It is necessary to further characterize HCV-6 genetics in this region.

Detection and Characterization of Homologues of Human Hepatitis Viruses and Pegiviruses in Rodents and Bats in Vietnam.

Rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. Numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. In this study of pegivirus and human hepatitis-related viruses, liver and serum samples from Vietnamese rodents and bats were examined by PCR and sequencing. Nucleic acids homologous to human hepatitis B, C, E viruses were detected in liver samples of 2 (1.3%) of 157 bats, 38 (8.1%), and 14 (3%) of 470 rodents, respectively. Hepacivirus-like viruses were frequently detected (42.7%) in the bamboo rat, Rhizomys pruinosus, while pegivirus RNA was only evident in 2 (0.3%) of 638 rodent serum samples. Complete or near-complete genome sequences of HBV, HEV and pegivirus homologues closely resembled those previously reported from rodents and bats. However, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the Hepacivirus genus. Of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined.

Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae.

We propose the creation of seven new species in the genus Pestivirus (family Flaviviridae) in addition to the four existing species, and naming species in a host-independent manner using the format Pestivirus X. Only the virus species names would change; virus isolates would still be referred to by their original names. The original species would be re-designated as Pestivirus A (original designation Bovine viral diarrhea virus 1), Pestivirus B (Bovine viral diarrhea virus 2), Pestivirus C (Classical swine fever virus) and Pestivirus D (Border disease virus). The seven new species (and example isolates) would be Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H (Hobi-like pestivirus), Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K (atypical porcine pestivirus). A bat-derived virus and pestiviruses identified from sheep and goat (Tunisian sheep pestiviruses), which lack complete coding region sequences, may represent two additional species.

Testing Convergent Evolution in Auditory Processing Genes between Echolocating Mammals and the Aye-Aye, a Percussive-Foraging Primate.

Several taxonomically distinct mammalian groups—certain microbats and cetaceans (e.g., dolphins)—share both morphological adaptations related to echolocation behavior and strong signatures of convergent evolution at the amino acid level across seven genes related to auditory processing. Aye-ayes (Daubentonia madagascariensis) are nocturnal lemurs with a specialized auditory processing system. Aye-ayes tap rapidly along the surfaces of trees, listening to reverberations to identify the mines of wood-boring insect larvae; this behavior has been hypothesized to functionally mimic echolocation. Here we investigated whether there are signals of convergence in auditory processing genes between aye-ayes and known mammalian echolocators. We developed a computational pipeline (Basic Exon Assembly Tool) that produces consensus sequences for regions of interest from shotgun genomic sequencing data for nonmodel organisms without requiring de novo genome assembly. We reconstructed complete coding region sequences for the seven convergent echolocating bat–dolphin genes for aye-ayes and another lemur. We compared sequences from these two lemurs in a phylogenetic framework with those of bat and dolphin echolocators and appropriate nonecholocating outgroups. Our analysis reaffirms the existence of amino acid convergence at these loci among echolocating bats and dolphins; some methods also detected signals of convergence between echolocating bats and both mice and elephants. However, we observed no significant signal of amino acid convergence between aye-ayes and echolocating bats and dolphins, suggesting that aye-aye tap-foraging auditory adaptations represent distinct evolutionary innovations. These results are also consistent with a developing consensus that convergent behavioral ecology does not reliably predict convergent molecular evolution.

Molecular characteristics and expression profile of the Rheb gene in Rongchang piglets (Sus scrofa)

To give some insights into the genetic functions of Rheb signaling pathway in Rongchang piglets, this study reported the identification of complete coding sequence of the Rheb gene in Rongchang piglets and indicated that the complete coding region sequence is 555bp in length and encodes 184 amino acids, containing nearly all the conserved protein domains in the Rheb protein family. The deduced amino acid sequence identity between the Rongchang pig and sheep, cattle, or goat is 100%, 99% and 98%, respectively. Spatial transcriptional profile analysis indicated that the Rheb gene is expressed at remarkably high levels in both salivary gland and brain, especially in hypothalamus and ventral hippocampus (VH) regions of brain. Developmental expression profile analysis revealed the expression pattern is different between brain sub-regions and salivary gland of these two breeds of piglets, with the similar expression pattern despite various mRNA levels between Rongchang and Large White piglets.

Sequence characterization and phylogenetic analysis of Toll-like receptor (TLR) 4 gene in the Tibetan macaque (Macaca thibetana).

In this study, the complete coding region sequence of an innate immune-related TLR4 gene was obtained from the Tibetan macaque (Macaca thibetana) genome via PCR and direct sequencing. The sequence had a total length of 2481 bp, contained 3 complete exons, and encoded 826 amino acids (AAs); its isoelectric point was 5.703, and the molecular weight was 94.72 kDa. The high structure prediction showed that the protein was comprised of one extracellular region, one transmembrane region, and one intracellular region. There were 48 potential functional sites in the protein, including glycosylation, phosphorylation, and acetylation sites. A homology analysis among 9 primate species, including the Tibetan macaque, human, chimpanzee, gibbon, rhesus macaque, cynomolgus monkey, pig-tailed monkey, squirrel monkey, and small-eared galago, showed that the homology of the nucleotide and AA sequences ranged from 60.9-99.5% and 51.4- 99.0%, respectively. Higher variability was identified in the extracellular region of the TLR4 protein, and its variable sites accounted for 88.79% (AA) of the total variable sites. Additionally, the number of AAs at the 3' end of the intracellular region was notably different among the primate lineages. The phylogenetic tree based on TLR4 gene exons of 9 primate species showed that the Tibetan macaque clustered with the rhesus macaque, cynomolgus monkey, and pig-tailed monkey; it was most distant from the small-eared galago. This study will provide an important basis for further study on the expression, regulation, and polymorphism of the TLR4 gene and the relationship between polymorphisms and host disease susceptibility.

Complete Coding Region Sequence of a Chikungunya Virus Strain Used for Formulation of CBER/FDA RNA Reference Reagents for Nucleic Acid Testing.

We have sequenced the complete coding region of a chikungunya virus isolate (R91064) used in the formulation of CBER/FDA RNA reference reagents for nucleic acid technology testing.

Genomic Characterization of Group C Orthobunyavirus Reference Strains and Recent South American Clinical Isolates

Group C orthobunyaviruses (family Bunyaviridae, genus Orthobunyavirus), discovered in the 1950s, are vector-borne human pathogens in the Americas. Currently there is a gap in genomic information for group C viruses. In this study, we obtained complete coding region sequences of reference strains of Caraparu (CARV), Oriboca (ORIV), Marituba (MTBV) and Madrid (MADV) viruses, and five clinical isolates from Peru and Bolivia, using an unbiased de novo approach consisting of random reverse transcription, random anchored PCR amplification, and high throughput pyrosequencing. The small, medium, and large segments encode for a 235 amino acid nucleocapsid protein, an approximately 1430 amino acid surface glycoprotein polyprotein precursor, and a 2248 amino acid RNA-dependent RNA polymerase, respectively. Additionally, the S segment encodes for an 83 amino acid non-structural protein, although this protein is truncated or silenced in some isolates. Phylogenetically, three clinical isolates clustered with CARV, one clustered with MTBV, and one isolate appeared to be a reassortant or a genetic drift resulted from the high variability of the medium segment which was also seen in a few other orthobunyaviruses. These data represent the first complete coding region sequences for this serocomplex of pathogenic orthobunyaviruses. The genome-wide phylogeny of reference strains is consistent with the antigenic properties of the viruses reported in the original serological studies conducted in the 1960s. Comparative analysis of conserved protein regions across group C virus strains and the other orthobunyavirus groups revealed that these group C viruses contain characteristic domains of potential structural and functional significance. Our results provide the basis for the developments of diagnostics, further genetic analyses, and future epidemiologic studies of group C viruses.

Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource

The 2005 consensus proposal for the classification of hepatitis C virus (HCV) presented an agreed and uniform nomenclature for HCV variants and the criteria for their assignment into genotypes and subtypes. Since its publication, the available dataset of HCV sequences has vastly expanded through advancement in nucleotide sequencing technologies and an increasing focus on the role of HCV genetic variation in disease and treatment outcomes. The current study represents a major update to the previous consensus HCV classification, incorporating additional sequence information derived from over 1,300 (near-)complete genome sequences of HCV available on public databases in May 2013. Analysis resolved several nomenclature conflicts between genotype designations and using consensus criteria created a classification of HCV into seven confirmed genotypes and 67 subtypes. There are 21 additional complete coding region sequences of unassigned subtype. The study additionally describes the development of a Web resource hosted by the International Committee for Taxonomy of Viruses (ICTV) that maintains and regularly updates tables of reference isolates, accession numbers, and annotated alignments (http://talk.ictvonline.org/links/hcv/hcv-classification.htm). The Flaviviridae Study Group urges those who need to check or propose new genotypes or subtypes of HCV to contact the Study Group in advance of publication to avoid nomenclature conflicts appearing in the literature. While the criteria for assigning genotypes and subtypes remain unchanged from previous consensus proposals, changes are proposed in the assignment of provisional subtypes, subtype numbering beyond “w,” and the nomenclature of intergenotypic recombinant. Conclusion: This study represents an important reference point for the consensus classification of HCV variants that will be of value to researchers working in clinical and basic science fields. (Hepatology 2014;59:318-327)

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Background: There are few data on the molecular basis of Cystic Fibrosis (CF) in North Africa, probably due to under-diagnosis.Aim: This is the first study of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in the Libyan population.Subjects and methods: This study analysed the complete coding region and flanking intronic sequences of the CFTR gene in 10 unrelated Libyan CF patients.Results: This study identified four mutations (F508del, c.1670delC, N1303K and E1104X), with a high frequency of the latter.Conclusion: Identification of CF mutations facilitates molecular investigation of cystic fibrosis in the Libyan population and helps to provide effective genetic counselling among CF families.

Cystic Fibrosis Transmembrane Conductance Regulator Mutation Spectrum in Patients with Cystic Fibrosis in Tunisia

To determine the frequency and types of mutations causing cystic fibrosis (CF) in Tunisia. We analyzed the complete coding region and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 68 unrelated patients suffering from the classical form of the disease. Twelve different CFTR mutations accounted for 90% (123/136) of CF alleles, including F508del (47.06%), E1104X (16.18%), N1303K (6.62%), 711 + 1T > G (5.88%), W1282X (4.41%), G542X (3.67%), R1158X (1.47%), 4016insT (0.74%), and R785X (0.74%). Three novel mutations were detected in this study: I1203V (1.47%), 1811 + 5A > G (0.74%), and 4268 + 2T > G (1.47%). Fifty patients (74%) were homozygous, among which 28 (41.17%) for F508del and 10 (14.7%) for E1104X. Ninety-seven percent of patients were found with at least one CFTR mutation. This study contributes to a better knowledge on CF-causing mutations in different regions in Tunisia and demonstrates that a complete scanning of CFTR sequences is necessary to implement efficient programs for CF genetic screening and counseling in this part of North Africa.

Multiple rare SAPAP3 missense variants in trichotillomania and OCD

Multiple rare SAPAP3 missense variants in trichotillomania and OCD

Evidence of Intragenic Recombination in G1 Rotavirus VP7 Genes

The G1 rotavirus is the most widespread genotype causing acute gastroenteritis in children. In an attempt to investigate the occurrence of intragenic recombination, 131 complete coding region sequences of VP7 genes of the G1 rotaviruses in GenBank were examined. Three hitherto-unreported intragenic recombinant rotaviruses were identified. It was noteworthy that two different types (interlineage and intersublineage) of intragenic recombination in rotaviruses were also found. This is the first report to demonstrate the existence of intragenic recombinations between interlineage and intersublineage in G1 rotaviruses.

A Y-linked SNP in SRY Gene Differentiates Chinese Indigenous Swamp Buffalo and Introduced River Buffalo

The complete coding region sequence of the SRY gene in Chinese swamp buffalo was determined by PCR product sequencing. Comparison of swamp and river buffalo SRY gene sequences revealed a single nucleotide polymorphism (SNP, C/G) at the 202 bp site of the coding region. Further, a total of 124 male domestic buffaloes were genotyped at this SNP site using the PCR-SSCP method, and it was found that all Chinese indigenous swamp buffaloes had a guanine (G) at this site, while introduced river buffaloes and crossbred buffaloes showed a cytosine (C). Our findings suggested that this Y-linked SNP displayed type-specific alleles differentiating swamp and river buffaloes, and could be used as an effective marker to detect crossbreeding of swamp buffaloes with introduced river buffaloes in native buffalo populations, and thereby assess genetic diversity status and make proper conservation decisions for indigenous swamp buffaloes. In addition, this SNP can be potentially applied in the study of Asian water buffalo phylogeny from a male perspective.

MtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences

The mitochondrial genome, contained in the subcellular mitochondrial network, encodes a small number of peptides pivotal for cellular energy production. Mitochondrial genes are highly polymorphic and cataloguing existing variation is of interest for medical scientists involved in the identification of mutations causing mitochondrial dysfunction, as well as for population genetics studies. Human Mitochondrial Genome Database (mtDB) () has provided a comprehensive database of complete human mitochondrial genomes since early 2000. At this time, owing to an increase in the number of published complete human mitochondrial genome sequences, it became necessary to provide a web-based database of human whole genome and complete coding region sequences. As of August 2005 this database contains 2104 sequences (1544 complete genome and 560 coding region) available to download or search for specific polymorphisms. Of special interest to medical researchers and population geneticists evaluating specific positions is a complete list of (currently 3311) mitochondrial polymorphisms among these sequences. Recent expansions in the capabilities of mtDB include a haplotype search function and the ability to identify and download sequences carrying particular variants.

The molecular epidemiology of dengue virus serotype 4 in Bangkok, Thailand

Dengue represents a major public health problem in Thailand, with all four viral serotypes co-circulating. Dengue virus serotype 4 (DENV-4) is the least frequently sampled serotype, although one that is often associated with hemorrhagic fever during secondary infection. To determine the evolutionary forces shaping the genetic diversity of DENV-4, and particularly whether its changing prevalence could be attributed to instances of adaptive evolution in the viral genome, we undertook a large-scale molecular epidemiological analysis of DENV-4 in Bangkok, Thailand, using both E gene and complete coding region sequences. This analysis revealed extensive genetic diversity within a single locality at a single time, including the discovery of a new and divergent genotype of DENV-4, as well as a pattern of continual lineage turnover. We also recorded the highest average rate of evolutionary change for this serotype, at 1.072 × 10 −3 nucleotide substitutions per site, per year. However, despite this abundant genetic variation, there was no evidence for adaptive evolution in any gene, codon, or lineage of DENV-4, with the highest rate of nonsynonymous substitution observed in NS2A. Consequently, the rapid turnover of DENV-4 lineages through time is most likely the consequence of a high rate of deleterious mutation in the viral genome coupled to seasonal fluctuations in the size of the vector population.