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Sclerotinia sclerotiorum reovirus 1 (SsReV1) was previously reported to infect hypovirulent strain SCH941 of the phytopathogenic fungus Sclerotinia sclerotiorum and to contain 11 double-stranded RNA (dsRNA) segments (S1-S11). Here, we report that SsReV1 is actually composed of 12 dsRNA segments instead of 11. The full-length nucleotide sequence of the twelfth segment (S12) was determined using a combination of RACE and high-throughput sequencing methods. S12 is 1217 nucleotides in length and has highly conserved terminal sequences that resemble those of the other 11 segments of SsReV1. S12 contains a single open reading frame encoding a protein (VP12) of 311 amino acids. Although regular BLAST analysis did not reveal any similarity of VP12 to known sequences, it was found to be homologous to the VP11 of Colorado tick fever virus of the genus Coltivirus when a hidden-Markov-model-based HHpred analysis was performed. A single-protoplast regeneration experiment suggested that S12 and S2 were maintained or lost in parallel. In summary, the SsReV1 genome consists of 12 dsRNA segments.

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus (CNSV), apple stem grooving virus (ASGV), lychnis mottle virus (LycMoV), grapevine line pattern virus (GLPV), and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleaotides (nt) in length, comprising three open reading frames (ORFs) and shared 63.8-75.9% nucleotide sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57% and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length with five ORFs encoding the REP (ORF1), triple gene block (TGB, ORF2-4) and coat protein (CP, ORF5) proteins. Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus (GYMaV) within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

HIV-1 subtype/circulating recombinant form (CRF) distribution of HIV-1-positive specimens for evaluating HIV in vitro diagnostics (IVDs) were examined and compared with the HIV-1 epidemic in Japan. The nucleotide sequences of the gag-pol region of 173 plasma specimens (84, provided in 2007 and 89 in 2013-2015) were determined. HIV-1 subtype/CRF classification was performed based on the phylogenetic analyses of the sequences. The subtype/CRF distribution resulting in this study was similar to that of a previous epidemiological report. Three CRF02_AG and one unique recombinant form (URF), including subtype G and A regions, were observed in the 2013 and 2014 specimens, except in the 2007 specimens. The reference panel consisting of these specimens was practical for the evaluation of HIV IVDs in Japan.

Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli. The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met1-Ala23), a catalytic domain (Val83-Glu347 and the catalytic triad Glu149, Glu171, and Ser218), a proline-rich hinge region (Pro414 -Pro450), a polycystic kidney disease protein motif (Gly 465-Ser 533), a C-terminus chitin-binding domain (Ala553- Glu593), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC–MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1). Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization.

Grapevine fabavirus (GFabV) was identified in the ‘Xiangfei’ grapevine in Liaoning, China, by small RNA sequencing. The complete nucleotide sequences of genomic RNA1 and RNA2 of the XF isolate were determined. The sizes of genomic RNA1 and RNA2 of XF isolate were 5 822 and 3 134 nucleotides (nt) respectively. Compared to other available GFabV genomes, XF isolate showed 81.7 ~82.7% and 84.8 ~94.9% in RNA1 polyprotein gene, and 67.2 ~82.8% and 78.0 ~96.1% in RNA2 polyprotein, at the nt and amino acid (aa) level, respectively. Phylogenetic analysis of complete and nearly complete genomic sequences of GFabV isolates showed XF form a separate evolutionary branch. This paper is the first report of the complete genome of GFabV in China.

Negeviruses are a group of insect-specific viruses that have a wide geographic distribution and broad host range. In recent years, nege-like viruses have been discovered in aphids of various genera of the family Aphididae, including Aphis, Rhopalosiphum, Sitobion, and Indomegoura. Here, we report the complete genome sequence of a nege-like virus isolated from Astegopteryx formosana aphids collected in Guangdong, China, which we have designated as "Astegopteryx formosana nege-like virus" (AFNLV). AFNLV has a genome length of 10,107 nt (excluding the polyA tail) and possesses the typical conserved domains of negeviruses. These include a viral methyltransferase, an S-adenosylmethionine-dependent methyltransferase, a viral helicase, and an RNA-dependent RNA polymerase (RdRP) domain in open reading frame 1 (ORF1), a DiSB-ORF2_chro domain in ORF2, and a SP24 domain in ORF3. The genome of AFNLV shares the highest nucleotide sequence identity (74.89%) with Wuhan house centipede virus, identified in a mixture of barley aphids. As clearly revealed by RdRP-based phylogenetic analysis, AFNLV, together with other negeviruses and nege-like viruses discovered in aphids, formed a distinct "unclassified clade" closely related to members of the proposed genus "Sandewavirus" and the family Kitaviridae. In addition, small interfering RNAs (siRNAs) derived from AFNLV did not exhibit typical characteristics of virus-derived siRNAs processed by the host RNAi-based antiviral pathway. However, the extremely high abundance of viral transcripts (average read coverage 73,403X) strongly suggested that AFNLV might actively replicate in the aphid host. AFNLV described in this study is the first nege-like virus discovered in aphids of the genus Astegopteryx, which will contribute to future study of the co-evolution of nege/nege-like viruses and their host aphids.

The full-length nucleotide sequence and genome organization of a novel mycovirus designated as “Rhizoctonia fumigata partitivirus 1” (RfPV1) from Rhizoctonia fumigata AG-Ba strain C-314 Baishi was determined. The genome of RfPV1 consists of two double-stranded RNAs (dsRNAs): dsRNA1 (2003 bp) and dsRNA2 (1802 bp). Each of the two dsRNAs contains one open reading frame, coding for a putative RNA-dependent RNA polymerase and a coat protein, respectively. The 5′ untranslated regions (UTRs) of both dsRNAs were conserved, and the 3′-UTRs of the two dsRNAs had interrupted poly(A) tails, similar to other partitiviruses. Phylogenetic analysis indicated that RfPV1 is a new species in the genus Alphapartitivirus, family Partitiviridae.

Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169–23.880% for COI, 1.336–23.434% for 12S, and 7.897–71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.

We extend the popular Jukes-Cantor evolution model and calculate the probability of an orthologous nucleotide sequence set [a reference sequence (B1) stays with the other sequences (B-1)], where the sequence evolution [from a last common ancestral sequence (ɑ)] follows the (prospective) Poisson process with the overall event rate λ prorated among mutation types (nucleotide/codon substitution, insertion, and deletion) and sites along each sequence. The corresponding retrospective process (reversing the prospective process) facilitates developing algorithms to calculate the marginal probability [Pr(B1)] (Monte Carlo integration) and sample ɑ (given B1). We calculate probability Pr(B-1|ɑ) based on the identified events (during "ɑ→B-1") from pairwise sequence alignment to implement Pr(B-1|B1) calculation (Monte Carlo integration). Event queue sampling and probability magnifiers are used to improve the computational efficiency when the number of events is large. We finally test our procedure on both simulated and recently studied hexapod transcriptome data (Brandt et al.), where each asexual lineage pairs with its closest related sexual lineage. Rate estimates (for Phasmatodea and Zygentoma) and model comparison indicate that the asexual lineages likely mutate several times faster than their sexual relatives.

Societal Impact Statement Digitized molecular data are vital to numerous aspects of scientific research and genetic resource use. The Convention on Biological Diversity currently refers to this as “Digital Sequence Information” (DSI), a term not widely adopted by science and lacking a clear definition. There are concerns over the access to genetic resources and absence of benefit sharing by provider countries. Open access to DSI might exacerbate this, which is leading to increasing policy interventions and restricted access to genetic resources and DSI. We analyze current international debate and proposed solutions and provide case studies of DSI use producing tangible benefits for the provider countries and scientific research, demonstrating the importance of open access DSI to achieving conservation goals. Summary Substantial advances in DNA sequencing over the last decades hold great potential to enhance food security and sustainable use of global biodiversity, benefiting the world's poorest people. Digital Sequence Information (DSI) plays a crucial role in catalyzing research applications that can contribute to international societal and biodiversity conservation targets. However, benefit sharing relating to DSI is difficult to identify and hindered by the lack of clear international governance and legislation, which in turn has led to a reluctance to make DSI publicly and freely available. Critically, no precise definition exists under the Convention on Biological Diversity (CBD), the Nagoya Protocol (NP), or the International Treaty for Plant Genetic Resources for Food and Agriculture (ITPGRFA). The key difference between DSI and biological resources, for which access and use are highly regulated under those frameworks, is that information is nonphysical. Information can be replicated and used without movement of, or access to, physical specimens. Thus, regulating the use of DSI is extremely challenging and remains controversial. Here, we review the regulation of DSI and the possible future steps by the international community, in the context of the benefit‐sharing obligations of the CBD, NP, and ITPGRFA. We highlight how multilateral agreements work in practice and are a solution to this impasse. We provide case studies demonstrating how the Royal Botanic Gardens, Kew, and its collaborators address the uncertainty surrounding the use of DSI, illustrating tangible and equitable benefits that have arisen from such use. We conclude that open access to DSI is needed for scientific research and international policy.

In 2019, a new disease characterized by yellow mottling on leaves was observed on Metaplexis japonica (Thunb.) Makino plants in Liaoning, China. Using RNA sequencing and PCR with overlapping primers, the complete nucleotide sequence of the circular double-stranded DNA genome of a new virus similar to caulimoviruses in diseased samples was determined. The genome comprised 7733 base pairs and was predicted to encode six proteins. It had the closest relationship to a caulimovirus, strawberry vein banding virus, based on sequence comparisons and phylogenetic analysis. Species-level sequence divergences from caulimoviruses were also observed in the conserved genomic regions. A complete episomal viral genome sequence in symptomatic M. japonica plants was identified, and the name “metaplexis yellow mottle-associated virus” is proposed for this virus.

The complete genome of a new umbra-like virus from edible fig (Ficus carica) was identified by high-throughput sequencing. Based on its similarity to umbra-like virus genome sequences available in GenBank, the proposed name of this new virus is "fig umbra-like virus" (FULV). The genome of full-length FULV-1 consists of 3049 nucleotides organized into three open reading frames (ORFs). Pairwise comparisons showed that the complete nucleotide sequence of the virus had the highest identity (71.3%) to citrus yellow vein-associated virus (CYVaV). In addition, phylogenetic trees based on whole-genome nucleotide sequences and amino acid sequences of the RNA-dependent RNA polymerase showed that FULV forms a monophyletic lineage with CYVaV and other umbra-like viruses. Based on the demarcation criteria of the genus Umbravirus, and lack of two umbravirus ORFs, we propose that FULV is a putative new member of the umbra-like virus clade within the family Tombusviridae.

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a plant showing mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus was found by Nanopore sequencing, and the full DNA-A sequence was confirmed by Sanger sequencing. The highest nucleotide identity match of this DNA-A genome (2596 bases) was 81.65% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% of nucleotide identity (DNA-A), we propose it as a new member of the genus Begomovirus, named Tomato mottle leaf distortion virus.

Virus detection is essential for the production and propagation of virus-free plants, quarantine for germplasm exchange, and discovery of new diseases. Citrus leaf blotch virus (CLBV) was first time detected, using high-throughput sequencing (HTS), in apple commercial orchards in the Henan province of China. Two isolates of CLBV (CLBV-PY1 and CLBV-PY2) were identified, and their full-length genomes are 8784 and 8792 nucleotides, respectively. Pairwise alignment revealed that CLBV-PY1 shared the highest nucleotide sequence identity (81.7%) with CLBV-PY2 and CLBV-PY2 had the highest nucleotide sequence identity (82.9%) with the Actinidia isolate HZ (accession No. MH427034). Results from both pairwise alignment and phylogenetic analysis demonstrated that these two isolates were closely related to the Actinidia isolates.

Klebsiella oxytoca is a gram-negative bacterium. It is opportunistic in nature and causes hospital acquired infections. Subtractive proteomics and reverse vaccinology approaches were employed to screen out the best proteins for vaccine designing. Whole proteome of K. oxytoca strain ATCC 8724, consisting of 5483 proteins, was used for designing the vaccine. Total 1670 cytotoxic T lymphocyte (CTL) epitope were predicted through NetCTL while 1270 helper T lymphocyte (HTL) epitopes were predicted through IEDB server. The epitopes were screened for non-toxicity, allergenicity, antigenicity and water solubility. After epitope screening 300 CTL and 250 HTL epitopes were submitted to IFN-γ epitope server to predict their Interferon-γ induction response. The selected IFN-γ positive epitopes were tested for their binding affinity with MHCI-DRB1 by MHCPred. The 15 CTL and 13 HTL epitopes were joined by linkers AAY and GPGPG respectively in vaccine construct. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine construct as an adjuvant. A 450aa long vaccine construct was submitted to I-TASSER server for 3D structure prediction. Thirteen Linear B cells were predicted by ABCPred server and 10 sets of discontinues epitopes for 3D vaccine structure were predicted by DiscoTope server. The modeled 3D vaccine construct was docked with human Toll-like receptor 2 (PDB ID: 6NIG) by PatchDock. The docked complexes were refined by FireDock. The selected docked complex showed five hydrogen bonds and one salt bridge. The vaccine sequence was reverse transcribed to get nucleotide sequence for In silico cloning. The reverse transcribed sequence strand was cloned in pET28a(+) expression vector. A clone containing 6586 bp was constructed including the 450 bp of query gene sequence.

To guarantee food safetyand sustainability, it is necessary to verify meat authenticity. This study focused on the development of single nucleotide polymorphism-based polymerase chain reaction-restriction fragment length polymorphism (SNP-based PCR-RFLP) and forensically informative nucleotide sequence (FINS) methodologies based on PCR amplification of the mitochondrial 12S rRNA gene for discrimination of six red meat species, that is, cattle, buffalo, goat, sheep, camel, and donkey. FINS allowed the unambiguous identification of all species analyzed. In addition, six SNPs, where a restriction site for TasI could be localized using a preliminary in silico analysis, gave a unique RFLP pattern for each species. The results revealed a low level of species substitution (8%) in the tested meat samples. In particular, one buffalo and goat samples have been substituted with cow and sheep, respectively. Finally, the developed techniques herein showed high potentials to be routinely used as reliable and fast tools to avoid meat species substitutions. PRACTICAL APPLICATION: This research deals with genetic techniques to trace meats. This kind of research helps the concerned agencies to build capacity to safeguard consumer sentiments as well as providing better market access and better food price and quality for the consumer.

Rhipicephalus microplus is the main blooding-sucking ectoparasite of bovines and is regarded as important vectors of animal diseases such as Babesiosis. Mining protective antigens of R. microplus to develop antitick vaccine is the most potential tick control strategy. In this study, the specific primers were designed according to the conserved nucleotide sequence of enolase gene in Haemaphysalis flava, Ixodes ricinus, and Ornithodoros moubata. The fragment of enolase gene was obtained by PCR using cDNA template from fully engorged female R. microplus. The full-length cDNA of enolase gene was amplified using rapid amplification of cDNA ends (RACE). Expression pattern of enolase gene in different tissues of R. microplus was analyzed by real-time quantitative PCR (qRT-PCR). Results showed that the full-length enolase cDNA containing 2052 bp was obtained successfully. The complete cDNA included an ORF of 1305 nucleotides encoding a protein of 434 amino acids. The enolase exhibited 85.0% amino acid identity to the enolase of H. flava, 81.1% to I. ricinus enolase, and 81.3% to O. moubata enolase. qRT-PCR analysis indicated that the enolase had the highest expression in the salivary gland of R. microplus.

A novel virus, Paris virus 2 (ParV2), was isolated from diseased Paris polyphylla var. yunnanensis, and its complete genome sequence was determined and analyzed. ParV2 is a positive-sense single-stranded RNA (+ssRNA) virus with a genome size of 4,118 nucleotides. The ParV2 genome contains six putative open reading frames (ORFs) that encode proteins with predicted molecular weights of 40.14, 100.26, 7.31, 7.85, 26.09, and 8.77 kDa. The first ORF (ORF1) of ParV2 encodes a putative protein of 40.14 kDa (P40, nt: 20-1,096), whiles the second ORF (ORF2, 888 aa) containing the GDD motif encodes the highly conserved RNA-dependent RNA polymerase protein (RdRP, nt:20-2,683, P100, 100.26 kDa) of viruses in the family Tombusviridae. Multiple sequence alignments analysis showed that the complete genome sequence of ParV2 shares 31.7-55.5% nucleotide sequence identities with viruses in the family Tombusviridae. Ginger chlorotic fleck-associated tombusvirus (GCFaV-1, Accession No. QKE30557) had the highest sequence identity (55.5%) with ParV2. GCFaV-1 also shares 59.2% RdRP and 34.9% CP amino acid sequence identities with ParV2. Sequence comparisons and phylogenetic analysis of RdRP suggested that ParV2 is a novel member of the family Tombusviridae, and its closest known relative is GCFaV-1.

Golden trumpet (Allamanda cathartica) plants were observed to exhibit mottling and distortion symptoms on leaves. The genome of an associated begomovirus (Al-K1) was amplified by rolling-circle amplification, cloned, and sequenced. The viral genome consisted of two circular ssDNA molecules, and the organization of the ORFs was similar to those of DNA-A and DNA-B components of bipartite begomoviruses. The size of DNA-A (KC202818) and DNA-B (MG969497) of the begomovirus was 2772 and 2690 nucleotides, respectively. Sequence analysis revealed that the DNA-A and DNA-B components shared the highest sequence identity with duranta leaf curl virus (MN537564, 87.8%) and cotton leaf curl Alabad virus (MH760452, 81.0%), respectively. Interestingly, the Al-K1 isolate shared significantly less nucleotide sequence identity with allamanda leaf curl virus (EF602306, 71.6%), the only monopartite begomovirus reported previously in golden trumpet from China. Al-K1 shared less than 91% sequence identity with other begomoviruses, and hence, according to the latest ICTV guidelines for species demarcation of begomoviruses, Al-K1 is proposed to be a member of a new species, and we propose the name "allamanda leaf mottle distortion virus" (AllLMoDV-[IN-Al_K1-12]) for this virus. AllLMoDV was detected in various golden trumpet samples from different locations by PCR with specific primers based on the genome sequence determined in this study. Our study provides evidence of the occurrence of a new bipartite begomovirus in a perennial ornamental plant in India.

In the study presented here, the first complete genome sequence of Leek yellow stripe virus (LYSV) designated as isolate LYSV-AE65 from Southwest of Iran, was reported. The small RNA deep sequencing analysis showed that, the Iranian isolate has a full RNA genome of 10,142 nucleotide in length (Except for poly (A) tail) and it was shared 77.91–92.16% nucleotide (nt) and 83.62–96.35% amino acid (aa) sequences identities with other known LYSV isolates. The coat protein (CP) region showed 80.21–95.24% nucleotide identity to those of other isolates, while high degrees of nucleotide sequence identity with G77-LYSV isolate (MN059504) from China. Phylogenetic analysis based on full genome sequence of LYSV-AE65, showed the closest relationship with LYSV isolates from China, Australia, Spain and Mexico. Also, phylogenetic analysis of the 5´-untranslated region (UTR)-P1 gene sequences of 44 isolates, confirmed the formation of two main groups, N-type and S-type, in agreement with the previous studies. Isolate LYSV-AE65 was similar to the members of clade S and has two large deletions in P1 gene. Recombination analysis demonstrated that LYSV-AE65 was a recombinant with most part of its genome was derived from already reported LYSV isolates infecting allium species. To the best of our knowledge, this is the first report of complete genome sequencing of LYSV isolate infecting garlic through small RNA deep sequencing method in Iran. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13337-021-00733-z).

Here, we describe a novel mycovirus, tentatively designated as "Botryosphaeria dothidea mitovirus 3" (BdMV3), isolated from Botryosphaeria dothidea strain FJ, which causes pear ring rot disease in Fujian Province, China. The complete genome nucleotide sequence of BdMV3 is 2538 nt in length and contains a single 2070-nt open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 689 amino acids (aa) using the fungal mitochondrial genetic code. BLASTp analysis revealed that the RdRp of BdMV3 shares 28.91%-69.36% sequence identity (query sequence coverage more than 90%) with those of members of the genus Mitovirus, with the highest sequence identity of 69.36% and 68.79% to the corresponding RdRp aa sequences of Rhizoctonia solani mitovirus 10 and Macrophomina phaseolina mitovirus 4, respectively. Phylogenetic analysis based on RdRp aa sequences indicated that BdMV3 is a new member of the genus Mitovirus in the family Mitoviridae.

Background Enterovirus 71 (EV-A71) is a highly infectious pathogen associated with hand, foot and mouth disease, herpangina, and various neurological complications, so it is important for the early detection and treatment of EV-A71. An aptamer is a nucleotide sequence that screened in vitro by the technology named systematic evolution of ligands by exponential enrichment technology (SELEX). Similar to antibodies, aptamers can bind to the targets with high specificity and affinity. Besides, emerging aptamers have many advantages comparing with antibodies, such as ease of synthesis and modification, having a wide variety of target materials, low manufacturing cost and easy flexibility in amending. Therefore, aptamers are promising in virus detection and anti-virus therapy. Methods Aptamers were selected by SELEX. Specificity, affinity and second structure were used to characterize the selected aptamers. Chemiluminescence was adopted to build an aptamer-based detection method for EV-A71. Cytopathogenic effects trial, the level of intracellular EV-A71 RNA and protein expression were used to evaluate the antiviral effect of the selected aptamers. Results Three DNA aptamers with high specificity and affinity for EV-A71structual protein VP1 were screened out. A rapid chemiluminutesescence aptamer biosensor for EV-A71 detection was designed out. The selected aptamers could inhibit the RNA replication and protein expression of EV-A71 in RD cells and ameliorate the cytopathogenic effects. Conclusions The aptamers against EV-A71 have the potentiality to be applied as attractive candidates used for EV-A71 detection and treatment in the future.

Cholesterol oxidases (CHOXs) are flavin‐adenine dinucleotide‐dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family members to meet the demands of enzyme markets worldwide. Here, we report the cloning, heterologous expression, and structural modeling of the cholesterol oxidase of Acinetobacter sp. strain RAMD. The cholesterol oxidase gene was cloned and expressed in pGEM®‐T and pET‐28a(+) vectors, respectively, using a gene‐specific primer based on the putative cholesterol oxidase ORF of Acinetobacter baumannii strain AB030 (GenBank [gb] locus tag: IX87_05230). The obtained nucleotide sequence (1671 bp, gb: MK575469.2), translated to a protein designated choxAB (556 amino acids), was overexpressed as inclusion bodies (IBs) (MW ˜ 62 kDa) in 1 mm IPTG‐induced Escherichia coli BL21 (DE3) Rosetta cells. The optimized expression conditions (1 mm IPTG with 2% [v/v] glycerol and at room temperature) yielded soluble active choxAB of 0.45 U·mL−1, with 56.25‐fold enhancement. The recombinant choxAB was purified to homogeneity using Ni2+‐affinity agarose column with specific activity (0.054 U·mg−1), yield (8.1%), and fold purification (11.69). Capillary isoelectric‐focusing indicated pI of 8.77 for choxAB. LC‐MS/MS confirmed the IBs (62 kDa), with 82.6% of the covered sequence being exclusive to A. baumannii cholesterol oxidase (UniProtKB: A0A0E1FG24). The 3D structure of choxAB was predicted using the LOMETS webtool with the cholesterol oxidase template of Streptomyces sp. SA‐COO (PDB: 2GEW). The predicted secondary structure included 18 α‐helices and 12 β‐strands, a predicted catalytic triad (E220, H380, and N514), and a conserved FAD‐binding sequence (GSGFGGSVSACRLTEKG). Future studies should consider fusion to solubilization tags and switching to the expression host Pichia pastoris to reduce IB formation., The cholesterol oxidase (choxAB) encoding ORF (1671 bp & 556 a.a) of Acinetobacter sp. strain RAMD was cloned (pGEM®‐T vector) and overexpressed (pET‐28a(+) vector) in E. coli BL21(DE3) Rosetta cells as inclusion bodies for the first time. Optimized production for recombinant soluble choxAB yields active choxAB (0.45 U·mL−1). The purified recombinant choxAB (62 kDa) showed 0.054 U·mg−1 and 11.69‐fold purification.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

Molecular Characters of AB-FAR Gene 1 of Aphelenchoides besseyi from Five Rice Varieties Aphelenchoides besseyi is known as nematode species that causes seed-transmitted white shoot disease in rice. One important gene that has been reported to control the pathogenicity of A. besseyi is AB FAR-1. The study was conducted to determine the character of the AB FAR-1 gene of A. besseyi extracted from rice seeds. Nematode extraction was carried out using Baerman funnel method from the seeds of 5 rice varieties, i.e. “Ciherang”, “Inpari Sidenuk”, “Sintanur”, “Prime Hybrid” and “Pak Tiwi”. Total DNA extraction of nematodes was conducted using CTAB method, followed by amplification of the AB FAR-1 gene using specific primers FAR-F1/R1 and nucleotide sequence analysis. Specific DNA band of AB FAR-1 gene measuring 150 bp was successfully amplified from all nematode samples. Sequence analysis showed that the AB FAR-1 gene had the highest homology (92.5 – 100%) with the Genbank accession no. JQ686690.1, which is the AB FAR-1 gene of A. besseyi from China. Despite having high homology, there were some nucleotide differences in the AB FAR-1 gene samples of A. besseyi from "Ciherang", "Inpari Sidenuk" and "Prime Hybrid". Further phylogenetic tree analysis differentiated the AB FAR-1 A. besseyi gene into 2 groups, i.e. group 1 consisting of the AB FAR-1 gene A. besseyi from China, "Sintanur", "Prime Hybrids" and "Pak Tiwi" and group 2 gene AB FAR-1 A. besseyi from "Ciherang", and "Inpari Sidenuk".

Background: The understanding of the secondary structure of the class 1 integron coded protein is necessary to decipher potential drug target and also to infer evolutionary ancestry at the proteomic level. This study was therefore aimed at determining the secondary structure of class 1 integron-coded protein and also to provide information on their evolutionary ancestry. Methodology: Five different sequences of Citrobacter freundii with the following accession numbers; KP902625.1, KP902624.1, KP902623.1, KP901093.1 and KP902609.1 were obtained using nucleotide BLAST (http://blast. ncbi.nlm.nih.gov/Blast.cgi) and subjected to evolutionary analysis, pairwise distance calculation, secondary structure and neutrality test using MEGA explorer, Kimura 2 parameter, SOPMA tool and Tajima’s test respectively. Results: Results of the NCBI queries revealed significant identity with class 1 integron of the studied Citrobacter freundii. The nucleotide sequence alignment depicted several conserved regions with varying degree of transitions, transversions, insertions, and deletions while the amino acid sequences of the nucleotides showed 42 conserved sites among all the sequences. The secondary structure of the class 1 integron coded protein depicted significant representation of the random coil (43.74±3.24), alpha helix (25.69±6.29) and the extended strands (22.42±2.41) than the beta turns (8.15±1.12). The Tajima’s Neutrality test of five nucleotide sequences of Citrobacter freundii analyzed by considering the first, second and third codons as well as the non-coding regions revealed a total of 127 positions in the final datasets while the Tajima’s Neutrality test was estimated to be -0.1038. Conclusion: The study confirmed common evolutionary ancestor for the class 1 integron coded protein found in Citrobacter freundii. Our study also documents the higher representation of random coil, alpha helix and extended strands than the beta turns. The negative value of the Tajima’s neutrality test suggests higher levels of both low and high frequency polymorphisms thus indicating a decrease in the class 1 integron population size and balancing selection Keywords: Evolutionary, Protein structure, Class 1 integrons, Citrobacter freundii

The prevailing COVID-19 pandemic has drawn the attention of the scientific community to study the evolutionary origin of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). This study is a comprehensive quantitative analysis of the protein-coding sequences of seven human coronaviruses (HCoVs) to decipher the nucleotide sequence variability and codon usage patterns. It is essential to understand the survival ability of the viruses, their adaptation to hosts, and their evolution. The current analysis revealed a high abundance of the relative dinucleotide (odds ratio), GC and CT pairs in the first and last two codon positions, respectively, as well as a low abundance of the CG pair in the last two positions of the codon, which might be related to the evolution of the viruses. A remarkable level of variability of GC content in the third position of the codon among the seven coronaviruses was observed. Codons with high RSCU values are primarily from the aliphatic and hydroxyl amino acid groups, and codons with low RSCU values belong to the aliphatic, cyclic, positively charged, and sulfur-containing amino acid groups. In order to elucidate the evolutionary processes of the seven coronaviruses, a phylogenetic tree (dendrogram) was constructed based on the RSCU scores of the codons. The severe and mild categories CoVs were positioned in different clades. A comparative phylogenetic study with other coronaviruses depicted that SARS-CoV-2 is close to the CoV isolated from pangolins (Manis javanica, Pangolin-CoV) and cats (Felis catus, SARS(r)-CoV). Further analysis of the effective number of codon (ENC) usage bias showed a relatively higher bias for SARS-CoV and MERS-CoV compared to SARS-CoV-2. The ENC plot against GC3 suggested that the mutational bias might have a role in determining the codon usage variation among candidate viruses. A codon adaptability study on a few human host parasites (from different kingdoms), including CoVs, showed a diverse adaptability pattern. SARS-CoV-2 and SARS-CoV exhibit relatively lower but similar codon adaptability compared to MERS-CoV.

The present article reviews the diagnosis of Novel Corona Virus Disease (COVID-19) by using Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR). COVID-19 is a contagious disease caused by severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), which affects upper and lower respiratory tract. In present scenario COVID-19 is considered as global public health emergency by World Health Organization (WHO) so, the detection and prevention of this disease is vital to control this emergency. RT-PCR is a highly sensitive laboratory method for the detection of SARS-CoV-2. It is based on rapid diagnosis of SARS-CoV-2 by qualitative detection of its nucleic acid sequence. This test locates the primer and probe sets in different regions in the genome of SARS-CoV-2.

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

Objectives The purpose of this study is to use the second internal transcribed spacer (ITS-2) to determine the molecular characteristics of Sarcoptes scabiei in rabbits from several areas of East Java. Methods Collecting S. scabiei mites from rabbits with clinical signs of scabies; DNA extraction with minikit QIAamp DNA; polymerase chain reaction amplification; nucleotide sequence analysis; homology and phylogenetic tree using the Neighbor-Joining method in the program molecular evolutionary genetics analysis-7 (MEGA-7). Results Sequence analysis of ITS-2 S. scabiei from five regions in East Java showed an identity >91.23% with isolates from China (KX695125.1). The phylogenetic analysis of ITS-2 S. scabiei from Mojokerto rabbits has a close relationship with AB82977.1; Surabaya and Nganjuk rabbits are closely related to KX695125.1; while Sidoarjo and Pasuruan rabbits are closely related to EF514469.2. and AB369384.1. Conclusions The homology analysis of all samples showed identity of more than 91.23% with isolate China (KX695125.1). The sequences of ITS-2 gen of S. scabiei from rabbits in several areas were relatively close to S. scabiei obtain various hosts from National Centre for Biotechnology Information (NCBI) data.

The decay of faecal matter from a septic system causes the arousal of fungi in the surrounding soil. These fungi can cause diseases if there is sewage spillage containing untreated or improperly treated wastewaters. Molecular techniques of identification of fungi have shown to be more dependable than traditional methods of identifying fungal species. This study was carried out to identify the fungal species associated with soil obtained from sewage-impacted soil near a septic tank using both traditional cultural techniques and molecular method. Fungi associated with the soil samples were isolated using serial dilution and Potato Dextrose Agar (PDA) method. Deoxyribonucleic Acid (DNA) was extracted from the pure cultures of fungal isolates using Quick DNA Fungal/Bacterial Miniprep kit. Polymerase Chain Reaction (PCR) amplification of internal transcribed spacer (ITS) region of the fungal isolates was carried out using universal primer pair; ITS4 and ITS5. The PCR products were sequenced and the sequences were blasted against National Centre for Biotechnology Information database. The result of the nucleotide sequence analysis revealed the identity of the isolates as Trichoderma harzanium with 580 base pairs and Aspergillus welwitschiae with 560 base pairs. Sequences of the isolates were aligned and compared with sequences on GenBank and a phylogenetic tree was constructed. The cultural method only aided in suggesting the suspected genera of the isolates while the molecular method was able to identify the isolates to the species level. This study will promote the knowledge of the fungal species associated with sewage-impacted soil and also aid researchers in proffering ways to enhance the prevention/control of diseases associated with sewage spill. Keyword: Septic tank, fungi, soil, phylogeny, sequencing

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function.

The Japanese rhinoceros beetle (Trypoxylus dichotomus [Allomyrina dichotoma]) produces the lectins allo A-I and allo A-II, which have strong N-acetyllactosamine (Galβ1-4GlcNAc)-binding activity. It has been suggested that the two lectins are formed from three subunits (α, β, and γ), with allo A-I comprising α and γ subunits and allo A-II comprising β and γ subunits. Here, we determined the cDNA sequences of these subunits using both conventional polymerase chain reaction (PCR)-cloning-sequencing and transcriptome-sequencing analyses. For the α and β subunits, one gene (locus) for each was predicted, whereas for the γ subunit, two types of cDNA sequences were obtained, which we named γ1 and γ2. These two types probably have distinct loci. Average nucleotide sequence identities among the subunits ranged from 87.6% (between α and γ1) to 92.6% (between γ1 and γ2), suggesting that they form a gene family. Although no homology was found between the sequences of allo A and other known lectin proteins in a protein database search, some unknown proteins containing the DUF3421 domain were identified. Those DUF3421 domain-encoding proteins are upregulated in the insect larval midgut. Thus, we infer that allo A genes also play an important role in larvae and that their lectin activity may have been obtained collaterally.

Lysine demethylase 3B (KDM3B) gene is a histone demethylase, demonstrating specific demethylation of the histone H3 lysine 9. It was detected as a sheep reproductive candidate gene by genome-wide s...

Genomoviridae is a virus family belonging to circular replication associated protein encoding ssDNA (CRESS-DNA) viruses, which have diverse genomic architecture and are widely distributed among different ecosystems. In this study, we characterized 39 novel genomovirus genomes including 3 from wild egrets, 9 from wild cranes, as well as 27 from wild finches in three different types of cloacal swabs of wild bird sampled in the station of Xinqing bird ringing, Maoershan Nature Reserve in Heilongjiang Province, Changbai Mountain in Jilin Province, and Hangzhou Wetland Park in Zhejiang Province, China. Here, using a Rep sequence phylogeny-based analysis, we divided the 39 genomoviruses into 8 genera within the family Genomoviridae, including Gemycircularvirus (n=20), Gemykibivirus (n=3), Gemygorvirus (n=2), Gemyvongvirus (n=2), Gemykolovirus (n=3), Gemykrogvirus (n=6), Gemytondvirus (n=1), Gemyduguivirus (n=1) and one unclassified genomovirus. The 39 genomovirus genomes belong to 36 species (27 of which are new) based on the currently accepted genomovirus pairwise nucleotide sequence identity species demarcation threshold of 78%. Overall, the research enriches our knowledge of CRESS-DNA viral diversity in China and emphasizes the prevalence of genomoviruses in nature.

Site-specific recombination (SSR) is an important tool in genome editing and gene circuit design. However, its applications are limited by the inability to simply and predictably tune SSR reaction rates across orders of magnitude. Facile rate manipulation can in principle be achieved by modifying the nucleotide sequence of the DNA substrate of the recombinase, but the design principles for rationally doing so have not been elucidated. To enable predictable tuning of SSR reaction kinetics via DNA sequence, we developed an integrated experimental and computational method to parse individual nucleotide contributions to the overall reaction rate, which we used to analyze and engineer the DNA attachment sequence attP for the inversion reaction mediated by the serine recombinase Bxb1. A quantitative PCR method was developed to measure the Bxb1 reaction rate in vitro. Then, attP sequence libraries were designed, selected, and sequenced to inform a machine-learning model, which revealed that the Bxb1 reaction rate can be accurately represented assuming independent contributions of nucleotides at key positions. Next, we used the model to predict the performance of DNA site variants in reaction rate assays both in vitro and in Escherichia coli, with flipping rates ranging from 0.01- to 10-fold that of the wild-type attP sequence. Finally, we demonstrate that attP variants with predictable DNA recombination rates can be used in concert to achieve kinetic control in gene circuit design by coordinating the coexpression of two proteins in both their relative proportion and their total amount. Our high-throughput, data-driven method for rationally tuning SSR reaction rates through DNA sequence modification enhances our understanding of recombinase function and expands the synthetic biology toolbox.Graphical abstract

Lumpy skin disease was first reported in the western provinces of Iran in 2014, and this was followed by several outbreaks throughout the country. In this study, 10 Iranian lumpy skin disease virus (LSDV) samples collected during the period of 2014-2018 were characterized by sequence analysis of the GPCR, LSDV142, and IL10LP genes. Sequence comparison of the respective genes revealed a close relationship between Iranian LSDV isolates and viruses from Asia and Europe. Interestingly, some nucleotide sequence diversity was also observed in the IL10LP genes of the Iranian field isolates.

Trichophyton equinum is a zoophilic dermatophyte that is frequently isolated from horse dermatophytosis and rare infections in humans. In the present study, molecular and physiological testing were performed on T. equinum isolates from dermatophytoses of Japanese racehorses to assess genotype and phenotype patterns of these strains. Comparative nucleotide sequence analysis showed that internal transcribed spacer (ITS) region sequences amplified from all Japanese isolates were 99.5% identical to T. equinum reference strains. ITS sequences amplified among the isolates were 100% (BT2) showed that isolates were 100% identical and harbored a "T" single nucleotide polymorphism at position 18. The sequences of β-tubulin (BT2) showed that isolates were 100% identical to T. equinum reference strains. The MAT1-2 allele was detected by PCR in all seven isolates, whereas none of the isolates contained the MAT1-1 allele. All isolates grew only on Trichophyton Agar 5 and did not grow on Trichophyton Agar 1 and 4, indicating nicotinic acid requirement. These results suggest that Japanese T. equinum isolates are derived from a clonal population.

A new member of the genus Closterovirus was detected in Platycodon grandiflorus using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named “platycodon closterovirus 1” (PlaCV1), comprises 16,771 nucleotides with nine predicted open reading frames (ORFs) having the typical closterovirus genome organization. PlaCV1 shares 37%–50% nucleotide sequence identity with other known closterovirus genome sequences. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), viral heat shock protein 90-like protein (HSP90h), minor coat protein (CPm), and coat protein (CP) show 47–68%, 39–66%, 24–52%, 21–57%, and 16–35% amino acid sequence identity, respectively, to homologous proteins in previously identified closteroviruses, suggesting that it represents a distinct, new species in the genus. Phylogenetic analysis of HSP70h sequences places PlaCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of PlaCV1 infecting P. grandiflorus in the Republic of Korea.

An educational activity is proposed that uses software for proteomic analysis and databases available for free on the Internet to estimate the structure and function of a hypothetical protein from its coding nucleotide sequence. This bioinformatics activity is recommended for integrated introductory courses that address the structure function relationship in proteins.