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In the present study we report the identification of a novel partitivirus recovered from Miscanthus sinensis, for which the provisional name "silvergrass cryptic virus 1" (SgCV-1) is proposed. High-throughput sequencing (HTS) and rapid amplification of cDNA ends (RACE) allowed the assembly of the complete sequence of each double-stranded RNA genome segment of this novel virus. The largest dsRNA segment, dsRNA1 (1699 bp), was predicted to encode a viral RNA-dependent RNA polymerase protein (RdRp) with 478 aa, and dsRNA2 (1490 bp) and dsRNA3 (1508 bp) were predicted to encode putative capsid proteins (CPs) with 347 and 348 aa, respectively. SgCV-1 has the highest amino acid sequence identity (≤ 70.80% in RdPp and ≤ 34.5% in CPs) to members of the genus Deltapartitivirus, family Partitiviridae, especially to unclassified viruses related to members of this genus. Its genome segment and protein lengths are also within the range of those of deltapartitiviruses. Moreover, phylogenetic analysis based on RdRp amino acid sequences also showed clustering of this novel virus with the related unclassified deltapartitiviruses. An RT-PCR survey of 94 imported M. sinensis samples held in quarantine identified seven additional samples carrying SgCV-1. This new virus fulfils all ICTV criteria to be considered a new member of the genus Deltapartitivirus.

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.

The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64 839 bp revealed 21 protein-coding genes and several hypothetical open reading frames with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related to the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in noncoding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.

In the summers of 2019 and 2020, a previously undescribed disease occurred in both juvenile and adult shellfish, causing mass mortalities in cultured pearl production, characterized by the major symptom of extreme atrophy of the soft tissues, including the mantle. However, the causative organism was uncertain. We isolated Vibrio sp. strain MA3 from the mantles of diseased pearl oysters Pinctada fucata. Analyses of 16S rRNA gene and DNA gyrase sequence homologies and its biochemical and morphological characteristics suggested that strain MA3 is a new strain of Vibrio alginolyticus. In addition, a hemolysin gene (Vhe1) of strain MA3 was detected as one of the virulence factors, and the complete sequence was determined. BLAST searches showed that Vhe1 shares 99.8% nucleotide sequence identity with Vibrio alginolyticus strain A056 lecithin-dependent hemolysin (ldh) gene, complete cds. Experimental infection of healthy oysters via injection with strain MA3 indicated it could cause high mortalities of the typically affected oysters from which the strain was isolated. These results suggest that the newly isolated Vibrio sp. strain MA3 is a putative causal agent of the recent disease outbreaks in Akoya pearl oysters.

Gitelman syndrome (GS) is an autosomal recessive disorder caused by loss-of-function mutations in SLC12A3, which encodes the Na-Cl cotransporter (NCC). Osteogenesis imperfecta (OI) is an autosomal dominant disorder caused by the inheritance of mutations mainly in the COL1A1 gene, resulting in bone fragility and deformity. In this study, we aimed to investigate the clinical and genetic manifestations in a 7-year-old boy with OI, who had electrolyte abnormalities and his four family members. Complete sequence analysis of COL1A1 revealed a novel mutation, c.268G>T, p.Glu90del. The gene mutation of OI in the patient's older brother was inherited from his mother, and the younger brother had no mutation. Two pathogenic mutations (c.179C>T, p.Thr60Met and c.1763C>T, p.Ala588Val) in SLC12A3 resulting in GS were also identified in the patient. The OI-related genetic mutation in the patient was consistent with that in the patient's mother. The GS-related genetic mutations were inherited from each parent. This study is the first to identify compound heterozygous variants in the SLC12A3 gene and a novel mutation in the COL1A1 gene in patients with OI and GS. Our findings indicate that genetic analysis is recommended to differentiate GS from BS, as clinical manifestations do not provide an accurate diagnosis.

The complete sequence of a new viral RNA from babaco (Vasconcellea × heilbornii) was determined. The genome consisted of 4,584 nucleotides, containing two open reading frames (ORFs 1 and 2), a 9-nt-long noncoding region (NCR) at the 5' terminus, and an unusually long (1,843 nt) NCR at the 3' terminus. The presence of a potential heptameric slippery signal located 12 nt upstream the stop codon of ORF 1 suggests a -1 ribosomal frameshift mechanism for the translation of ORF 2. Sequence comparisons of ORF 2 revealed similarity to the RNA-dependent RNA polymerase (RdRp) of several umbra- and umbra-like viruses. Phylogenetic analysis of the RdRp placed the new virus in a well-supported and cohesive clade that includes umbra-like viruses reported in papaya, citrus, opuntia, maize, and sugarcane hosts. Viruses of this clade share a most recent ancestor with the umbraviruses but have different genomic features. The creation of a new genus within the family Tombusviridae is proposed for the classification of these novel viruses.

The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEPs), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins, with a focus on SEPs, in the archaeon Methanosarcina mazei. Combining the data of all proteases, we identified 63 small proteins and additional 28 SEPs with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEPs, a complete sequence coverage was achieved. Moreover, for five SEPs, incorrectly predicted translation start points or potential in vivo proteolytic processing were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach allows to improve the identification and molecular characterization of small proteins and SEPs. LC-MS data: ProteomeXchange PXD023921.

Ornithogalum thyrsoides, a widely cultivated bulbous ornamental plant endemic to South Africa, has significant commercial value as a pot plant and for the production of cut flowers. However, infection by viruses threatens the success of commercial cultivation, as symptoms negatively affect the appearance of the plant and flowers. To date, four Ornithogalum-infecting viruses have been reported. Complete genome sequence data are available for three of these viruses, but the genome of the potyvirus ornithogalum virus 3 (OV3) has not been fully sequenced. In this study, the complete sequence of OV3 was determined by high-throughput sequencing (HTS) and validated by Sanger sequencing. Based on recognition of protease cleavage patterns and multiple sequence alignments with closely related viruses, the polyprotein of OV3 was predicted to be proteolytically cleaved to produce 10 mature peptides containing domains conserved in members of the genus Potyvirus. Phylogenetic analysis and species demarcation criteria confirm the previous classification of OV3 as a member of a separate species in this genus. This is the first report of a complete genome sequence of OV3.

This work introduces new morphological and molecular information on the filaroid nematodeSetaria cervi(Rudolphi, 1819) obtained from 13 infected game ungulates out of 96 dissected. The hosts comprised the following: a single moose (Alces alces), ten red deer (Cervus elaphus) and two sika deer (Cervus nippon) originating from the western and northern regions of the Czech Republic. Based on the complete sequences of the gene encoding mitochondrial cytochrome c oxidase subunit 1 (cox1), all 20 females and four males belonged to the speciesS. cervi. We detected three developmental female stages (adult fertile females, juvenile L5 females and L4 female larvae) differing in size and some morphological traits as the subtle structure of peribuccal crown and shape and features of tail knob. Such differences were described in detail for the first time. The phylogenetic relationships within the family Onchocercidae have been evaluated using new information on thecox1 sequence ofS. cervi(maximum likelihood method, GTR + I + G model). In accordance with the latest phylogenetic studies, the present analysis confirmed the ancient separation of the subclass Setariinae from the remaining two onchocercid lineages Dirofilariinae and Onchocerinae.

Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5' and 3' RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.

The complete sequence of the mitochondrial genome of Balanus trigonus Darwin, 1854 was examined using next-generation sequencing analysis. The complete mitogenome of B. trigonus has 15,336 bp in length and comprises 37 genes, namely, 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs. Both the gene order and characteristics are consistent with those of other species within the family Balanidae. Phylogenetic analysis based on complete mitogenomes revealed taxonomic relationships among members of the family Balanidae.

The complete genome sequence of a novel mycovirus, Phoma matteucciicola RNA virus 1 (PmRV1), derived from Phoma matteucciicola strain LG-01, was sequenced and analyzed. The complete cDNA sequence of PmRV1 is 3432 bp in length with a GC content of 57.17%. The genome of PmRV1 contains two putative open reading frames (ORFs): ORF1 and ORF2. ORF1 encodes a hypothetical protein with significant similarity to a protein encoded by Periconia macrospinosa ambiguivirus 1 (PmAV1). ORF2 encodes a protein of 491 amino acids with a conserved RNA-dependent RNA polymerase (RdRp) domain. Additionally, the triad within domain III has an asparagine (GDN) instead of the nearly universally conserved aspartic acid (GDD). RdRp phylogeny showed that PmRV1 grouped together with PmAV1 as a sister branch of a new member of the recently proposed family of mycotombus-like viruses. This is first report of the complete sequence of a novel mycovirus, PmRV1, infecting Phoma matteucciicola strain LG-01, the causal agent of leaf blight of Curcuma wenyujin.

Papaya ringspot virus (PRSV) is one of the important pathogens responsible for damage of the papaya crop and causes significant yield loss of fruits and vegetable worldwide and hence a serious threat. In this study, PRSV-infected plant samples collected from different locations in North 24 Parganas districts of West Bengal, were tested with potyvirus specific ELISA and further confirmed with potyvirus group specific degenerate primers by RT PCR, TEM and sequence analysis. Our result indicates that the disease symptoms occurred in 80–100% of the plant in the field surveyed. The complete sequence of PRSV isolate WB was determined from RT-PCR products with specific primer pairs designed covering the whole genome and confirmed by next-generation sequencing. The whole genome of the PRSV was determined and submitted to GenBank as accession no. LC482263. The sequence of PRSV-WB (10,340 bp) was found to share upto 90% and 93%, identity with other PRSV isolates at the nucleotide and amino acid levels respectively. Based on phylogenetic analysis, PRSV- WB was found to be most closely related to PRSV-BD2 (MH397222) from papaya in Bangladesh (geographically close).

Objective Amomum villosum (AV) is an herb whose dried fruit has been extensively used in modern medicine to treat digestive system diseases such as dysentery, vomiting and abdominal pain. This paper aims to supplement chloroplast (cp) genomic resources and to be used in phylogenetic studies and identification of AV related plants. Methods High-throughput sequencing technology was used to determine the complete sequence of the AV cp genome, and the sequence was then compared with three related species. Results The genome size of AV we obtained was 163,968 bp with an obvious tetrad structure. The AV cp genome was observed to contain 125 unique genes and 81 simple sequence repeat (SSRs) had been determined and the majority of which were adenine-thymine (AT)-rich. Comparative analysis of genome sequence of four ginger plants showed that the atpF, clpP and rpl32 genes are potential markers for identifying Amomum species. Phylogenetic analysis suggested that AV was closely related to A. kravanh and A. compactum. Conclusion These results have brought useful genetic resources for further identification researches, DNA barcoding, resolving taxonomy and understanding the evolutionary mode of Zingiberaceae cp genome.

Ancient DNA studies provide genomic information about the origins, population structures, and physical characteristics of ancient humans that cannot be solely examined by archeological studies. The DNAs extracted from ancient human bones, teeth, or tissues are often contaminated with coexisting bacterial and viral genomes that contain DNA from ancient microbes infecting those of ancient humans. Information on ancient viral genomes is useful in making inferences about the viral evolution. Here, we have utilized metagenomic sequencing data from the dental pulp of five Jomon individuals, who lived on the Japanese archipelago more than 3000 years ago; this is to detect ancient viral genomes. We conducted de novo assembly of the non-human reads where we have obtained 277,387 contigs that were longer than 1000 bp. These contigs were subjected to homology searches against a collection of modern viral genome sequences. We were able to detect eleven putative ancient viral genomes. Among them, we reconstructed the complete sequence of the Siphovirus contig89 (CT89) viral genome. The Jomon CT89-like sequence was determined to contain 59 open reading frames, among which five genes known to encode phage proteins were under strong purifying selection. The host of CT89 was predicted to be Schaalia meyeri, a bacterium residing in the human oral cavity. Finally, the CT89 phylogenetic tree showed two clusters, from both of which the Jomon sequence was separated. Our results suggest that metagenomic information from the dental pulp of the Jomon people is essential in retrieving ancient viral genomes used to examine their evolution.

We present here the first complete sequence of cucumber mosaic virus (CMV) from RNA sequencing of a dwarf periwinkle (Vinca minor) and a Wisteria sinensis in Iran by de novo assembly and annotation of contigs. The tripartite genome of CMV-Ir-VM and Ir-WS consists of a 3391-nucleotide (nt) RNA1, a 3038-nt RNA2, and a 2197-nt RNA3 segment. The sequence comparisons and phylogenetic analyses revealed that all three RNA segments of these two isolates belonged to the CMV subgroup II. RNA1, RNA2, and RNA3 of these two isolates, respectively shared 97.61–99.06%, 97.83–98.88%, and 97.19–98.86% nucleotide identities to those of CMV subgroup II isolates. In genetic analysis, the 2b gene revealed more genetic variability (π = 0.2614) compared with other regions. Our results indicated that purifying selection pressure was the major force acting upon the CMV genome, although some positively selected sites were identified for all encoded proteins.

Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS2 spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase). Then, based on the characteristics of peptide ladders, complete sequencing was realized by comparing MS2 spectral similarity of the generated peptide ladders. The complete amino acid sequences of bivalirudin, adrenocorticotropic hormone, and oxytocin were determined with high accuracy. This approach is beneficial to the quality control of drug peptides as well as the identification of novel bioactive peptides.

We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (NGS). The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety-seven of these samples were previously characterized at MICA by legacy methods (Sanger or sequence specific oligonucleotide) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology (ONT), on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with ONT, extending from the 5' untranslated region (UTR) to the 3' UTR of the MICA gene. Thirty-two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed.

Infection of bovine papillomavirus (BPV) has been associated with mucosal and/or cutaneous tumor development in bovids. To date, up to 27 genotypes of BPVs have been identified and classified based on the nucleotide sequence identity of L1 open reading frame. In the present study, the complete sequence of a novel BPV concurrently identified with BPV1 and BPV2 in the facial cutaneous papilloma lesion of a domestic cattle was characterized. The whole genome of the unclassified BPV was 7263 base pairs in full length with GC ratio of 42.9%. In comparison with published BPV sequences, L1 nucleotide sequence of the novel BPV shared 75% identity with BPV15, and was suggested to be classified in the genus, Xipapillomavirus. According to the criteria established by the International Committee on the Taxonomy of Viruses, the novel BPV was designated as BPV type 28.

Talaromyces is a monophyletic genus containing seven sections. The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, BenA, CaM and RPB2 gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other Talaromyces species are presented for these putative new species. Penicillium resedanum is combined in Talaromyces section Subinflati as T. resedanus.

Antibody-drug conjugates (ADCs) are an increasingly prevalent drug class utilized as chemotherapeutic agents. The complexity of ADCs, including their large size, array of drug conjugation sites, and heterogeneous compositions containing from zero to several payloads, demands the use of advanced analytical characterization methods. Tandem mass spectrometry (MS/MS) strategies, including a variety of bottom-up, middle-down, and even top-down approaches, frequently applied for the analysis of antibodies are increasingly being adapted for antibody-drug conjugates. Middle-down tandem mass spectrometry, often focusing on the analysis of ∼25 kDa protein subunits, offers the potential for complete sequence confirmation as well as the identification of multiple conjugation states. While middle-down studies have been extensively developed for monoclonal antibodies, middle-down characterization of ADCs has been limited by the high complexity of the drug molecules. This study seeks to bridge the gap by utilizing a combination of 193 nm ultraviolet photodissociation (UVPD), electron-transfer dissociation (ETD), and electron-transfer/higher-energy collision dissociation (EThcD). The compilation of these MS/MS methods leads to high sequence coverages of 60-80% for each subunit of the ADC. Moreover, the combined fragmentation patterns provide sufficient information to allow confirmation of both the sequence of the complementarity-determining regions and the payload conjugation sites.

Begomoviruses (family Geminiviridae, genus Begomovirus) are responsible for considerable losses in several tropical crops, more commonly in bean and tomato. Begomoviruses comprise the most numerous and economically destructive group within Geminiviridae. Characteristic symptoms of begomovirus infection in Capsicum chinense (chili pepper, Solanaceae) were observed in smallholder farms in the state of Amazonas. The molecular detection and identification of begomoviruses affecting chili pepper were conducted from symptomatic foliar samples collected at 14 farms in the municipalities of Presidente Figueiredo, Iranduba and Manaus (Amazonas state). Begomovirus was detected in five out of the 69 samples, all of the cultivar Lupita. Pairwise sequence comparisons and phylogenetic analysis based on the complete sequence of the DNA-A component identified the isolates as members of the species Euphorbia yellow mosaic virus (EuYMV). Comparative analysis with 36 EuYMV isolates grouped the Amazonian isolates in a single clade, indicating a low degree of genetic variability for this begomovirus.

Rice stripe virus (RSV) causes viral disease and yield losses in rice. Leaves of rice plants showing yellowing, stripes and curling were collected in Sichuan Province, China and tested for rice strip virus (RSV) by RT-PCR using specific primers. Nine of the nineteen symptomatic samples were positive for RSV. The complete sequence of RSV RNA1 to RNA4 was determined by assembling cDNA fragments obtained from isolate Huid04 in Huidong County, Sichuan Province (GenBank accession Nos. KX611337 to KX611340). Phylogenetic analyses of RNA-dependent RNA polymerase sequences showed a close relationship of RSV isolate Hui0d4 and isolates DaL08 and KunM08 from Yunnan Province, China. This is the first complete genomic sequence of an RSV isolate from Sichuan Province, China.

ObjectivesTo investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:−.MethodsA total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014–19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms.ResultsWe identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles.ConclusionsOur findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

We provide the complete sequence of a virus tentatively named "Tetranychus urticae-associated picorna-like virus 1PK13" (TuaPV1-PK13) obtained from the high-throughput sequencing of a symptomless apple leaf sample. Although the virus sequence was originally derived from apple leaves, the data suggest that the virus is associated with the two-spotted mite Tetranychus urticae.

We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.

In this study, we investigated the molecular characteristics and spatio-temporal dynamics of GII.P17-GII.17 norovirus in Zhoushan Islands during 2013–2018. We collected 1849 samples from sporadic acute gastroenteritis patients between January 2013 and August 2018 in Zhoushan Islands, China. Among the 1849 samples, 134 (7.24%) samples were positive for human norovirus (HuNoV). The complete sequence of GII.17 VP1 gene was amplified from 31 HuNoV-positive samples and sequenced. A phylogenetic tree was constructed based on the full-length sequence of the VP1 gene. Phylogenetic analysis revealed that the GII.17 genotype detected during 2014–2018 belongs to the new GII.17 Kawasaki variant. Divergence analysis revealed that the time of the most recent common ancestor (TMRCA) of GII.17 in Zhoushan Islands was estimated to be between 1997 and 1998. The evolutionary rate of the VP1 gene of the GII.17 genotype norovirus was 1.14 × 10–3 (95% HPD: 0.62–1.73 × 10–3) nucleotide substitutions/site/year. The spatio-temporal diffusion analysis of the GII.17 genotype identified Hong Kong as the epicenter for GII.17 dissemination. The VP1 gene sequence of Zhoushan Island isolates correlated with that of Hong Kong and Japan isolates.

We study generalized Fibonacci sequences $F_{n+1}=PF_n-QF_{n-1}$ with initial values $F_0=0$ and $F_1=1$. Let $P,Q$ be nonzero integers such that $P^2-4Q$ is not a perfect square. We show that if $Q=\pm 1$ then the sequence $\{F_n\}_{n=0}^\infty$ misses a congruence class modulo every prime large enough. On the other hand, if $Q \neq \pm 1$, we prove that (under GRH) the sequence $\{F_n\}_{n=0}^\infty$ hits every congruence class modulo infinitely many primes., 13 pages