Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

Rapid protein sequence evolution via compensatory frameshift is widespread in RNA virus genomes.

BACKGROUND: RNA viruses possess remarkable evolutionary versatility driven by the high mutability of their genomes. Frameshifting nucleotide insertions or deletions (indels), which cause the premature termination of proteins, are frequently observed in the coding sequences of various viral genomes. When a secondary indel occurs near the primary indel site, the open reading frame can be restored to produce functional proteins, a phenomenon known as the compensatory frameshift. RESULTS: In this study, we systematically analyzed publicly available viral genome sequences and identified compensatory frameshift events in hundreds of viral protein-coding sequences. Compensatory frameshift events resulted in large-scale amino acid differences between the compensatory frameshift form and the wild type even though their nucleotide sequences were almost identical. Phylogenetic analyses revealed that the evolutionary distance between proteins with and without a compensatory frameshift were significantly overestimated because amino acid mismatches caused by compensatory frameshifts were counted as substitutions. Further, this could cause compensatory frameshift forms to branch in different locations in the protein and nucleotide trees, which may obscure the correct interpretation of phylogenetic relationships between variant viruses. CONCLUSIONS: Our results imply that the compensatory frameshift is one of the mechanisms driving the rapid protein evolution of RNA viruses and potentially assisting their host-range expansion and adaptation.

A novel tepovirus, Agave virus T, identified by the analysis of the transcriptome data of blue agave (Agave tequilana).

The genome sequence of a novel RNA virus was identified by analyzing transcriptome data obtained from the stem sample of a blue agave (Agave tequilana) plant. Sequence comparison and phylogenetic analysis showed that the RNA virus, Agave virus T (AgVT), was a new member of the genus Tepovirus in the family Betaflexiviridae. AgVT genome had three open reading frames: a 1605-amino acid (aa) replicase (REP), 355-aa movement protein (MP), and 220-aa coat protein (CP). Phylogenetic analyses based on the REP, MP, and CP sequences of AgVT, previously reported tepoviruses, and other Betaflexiviridae viruses revealed that tepoviruses could be classified into two subclades: "potato virus T (PVT)-clade" and "Prunus virus T (PrVT)-clade." PVT, the type species and founding member of the genus Tepovirus, belong to "PVT-clade" along with AgVT, while the other five tepoviruses belong to "PrVT-clade." The genome sequence of AgVT may be useful for studying the phylogenetic relationships between tepoviruses and other closely related viruses. Keywords: Agave virus T; Tepovirus; Betaflexiviridae; blue agave; Agave tequilana.

Two novel closteroviruses, fig virus A and fig virus B, identified by the analysis of the high-throughput RNA-sequencing data of fig (Ficus carica) latex.

Closteroviruses (the genus Closterovirus, the family Closteroviridae) are RNA viruses that infect and cause viral diseases in many economically important plants. Genome sequences of two novel closteroviruses named fig virus A (FiVA) and fig virus B (FiVB) were identified in high-throughput sequencing data obtained from a fig latex sample. FiVA and FiVB genomes, whose lengths are 19,333 bp and 18,741 bp, respectively, were predicted to have 14 shared open reading frames, nine of which had homologs in other closteroviruses. Phylogenetic analysis confirmed that FiVA and FiVB are novel closteroviruses forming a strong subclade with fig mild mottle-associated virus within the genus Closterovirus. FiVA and FiVB genome sequences identified in this study are useful resources for investigating the evolution of closterovirus genome organization. Keywords: fig virus A; fig virus B; Closterovirus; common fig; Ficus carica.

Diet-Related Alterations of Gut Bile Salt Hydrolases Determined Using a Metagenomic Analysis of the Human Microbiome.

The metabolism of bile acid by the gut microbiota is associated with host health. Bile salt hydrolases (BSHs) play a crucial role in controlling microbial bile acid metabolism. Herein, we conducted a comparative study to investigate the alterations in the abundance of BSHs using data from three human studies involving dietary interventions, which included a ketogenetic diet (KD) versus baseline diet (BD), overfeeding diet (OFD) versus underfeeding diet, and low-carbohydrate diet (LCD) versus BD. The KD increased BSH abundance compared to the BD, while the OFD and LCD did not change the total abundance of BSHs in the human gut. BSHs can be classified into seven clusters; Clusters 1 to 4 are relatively abundant in the gut. In the KD cohort, the levels of BSHs from Clusters 1, 3, and 4 increased significantly, whereas there was no notable change in the levels of BSHs from the clusters in the OFD and LCD cohorts. Taxonomic studies showed that members of the phyla Bacteroidetes, Firmicutes, and Actinobacteria predominantly produced BSHs. The KD altered the community structure of BSH-active bacteria, causing an increase in the abundance of Bacteroidetes and decrease in Actinobacteria. In contrast, the abundance of BSH-active Bacteroidetes decreased in the OFD cohort, and no significant change was observed in the LCD cohort. These results highlight that dietary patterns are associated with the abundance of BSHs and community structure of BSH-active bacteria and demonstrate the possibility of manipulating the composition of BSHs in the gut through dietary interventions to impact human health.

Genomic and metabolic features of Tetragenococcus halophilus as revealed by pan-genome and transcriptome analyses.

The genomic and metabolic diversity and features of Tetragenococcus halophilus, a moderately halophilic lactic acid bacterium, were investigated by pan-genome, transcriptome, and metabolite analyses. Phylogenetic analyses based on the 16S rRNA gene and genome sequences of 15 T. halophilus strains revealed their phylogenetic distinctness from other Tetragenococcus species. Pan-genome analysis of the T. halophilus strains showed that their carbohydrate metabolic capabilities were diverse and strain dependent. Aside from one histidine decarboxylase gene in one strain, no decarboxylase gene associated with biogenic amine production was identified from the genomes. However, T. halophilus DSM 20339T produced tyramine without a biogenic amine-producing decarboxylase gene, suggesting the presence of an unidentified tyramine-producing gene. Our reconstruction of the metabolic pathways of these strains showed that T. halophilus harbors a facultative lactic acid fermentation pathway to produce l-lactate, ethanol, acetate, and CO2 from various carbohydrates. The transcriptomic analysis of strain DSM 20339T suggested that T. halophilus may produce more acetate via the heterolactic pathway (including d-ribose metabolism) at high salt conditions. Although genes associated with the metabolism of glycine betaine, proline, glutamate, glutamine, choline, and citrulline were identified from the T. halophilus genomes, the transcriptome and metabolite analyses suggested that glycine betaine was the main compatible solute responding to high salt concentration and that citrulline may play an important role in the coping mechanism against high salinity-induced osmotic stresses. Our results will provide a better understanding of the genome and metabolic features of T. halophilus, which has implications for the food fermentation industry.

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation.

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

The host protease KLK5 primes and activates spike proteins to promote human betacoronavirus replication and lung inflammation.

Coronaviruses rely on host proteases to activate the viral spike protein, which facilitates fusion with the host cell membrane and the release of viral genomic RNAs into the host cell cytoplasm. The distribution of specific host proteases in the host determines the host, tissue, and cellular tropism of these viruses. Here, we identified the kallikrein (KLK) family member KLK5 as a major host protease secreted by human airway cells and exploited by multiple human betacoronaviruses. KLK5 cleaved both the priming (S1/S2) and activation (S2') sites of spike proteins from various human betacoronaviruses in vitro. In contrast, KLK12 and KLK13 displayed preferences for either the S2' or S1/S2 site, respectively. Whereas KLK12 and KLK13 worked in concert to activate SARS-CoV-2 and MERS-CoV spike proteins, KLK5 by itself efficiently activated spike proteins from several human betacoronaviruses, including SARS-CoV-2. Infection of differentiated human bronchial epithelial cells (HBECs) with human betacoronaviruses induced an increase in KLK5 that promoted virus replication. Furthermore, ursolic acid and other related plant-derived triterpenoids that inhibit KLK5 effectively suppressed the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2 in HBECs and mitigated lung inflammation in mice infected with MERS-CoV or SARS-CoV-2. We propose that KLK5 is a pancoronavirus host factor and a promising therapeutic target for current and future coronavirus-induced diseases.

Deciphering the evolutionary landscape of severe fever with thrombocytopenia syndrome virus across East Asia.

Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a significant public health challenge in East Asia, necessitating a deeper understanding of its evolutionary dynamics to effectively manage its spread and pathogenicity. This study provides a comprehensive analysis of the genetic diversity, recombination patterns, and selection pressures across the SFTSV genome, utilizing an extensive dataset of 2041 sequences from various hosts and regions up to November 2023. Employing maximum likelihood and Bayesian evolutionary analysis by sampling trees (BEAST), we elucidated the phylogenetic relationships among nine distinct SFTSV genotypes (A, B1, B2, B3, B4, C, D, E, and F), revealing intricate patterns of viral evolution and genotype distribution across China, South Korea, and Japan. Furthermore, our analysis identified 34 potential reassortments, underscoring a dynamic genetic interplay among SFTSV strains. Genetic recombination was observed most frequently in the large segment and least in the small segment, with notable recombination hotspots characterized by stem-loop hairpin structures, indicative of a structural propensity for genetic recombination. Additionally, selection pressure analysis on critical viral genes indicated a predominant trend of negative selection, with specific sites within the RNA-dependent RNA polymerase and glycoprotein genes showing positive selection. These sites suggest evolutionary adaptations to host immune responses and environmental pressures. This study sheds light on the intricate evolutionary mechanisms shaping SFTSV, offering insights into its adaptive strategies and potential implications for vaccine development and therapeutic interventions.

Bactericidal Effect of Combination of Atmospheric Pressure Plasma and Nisin on Meat Products Inoculated with Escherichia coli O157:H7.

This study was conducted to investigate the bactericidal effect of nisin (Nisin) only, atmospheric pressure plasma (APP) only, and a combination of APP and nisin (APP+Nisin) on beef jerky and sliced ham inoculated with Escherichia coli O157:H7, gram-negative bacteria. The bactericidal effect against E. coli O157:H7 and Listeria monocytogenes was confirmed using a nisin solution at a concentration of 0-100 ppm, and APP+Nisin was tested on beef jerky and sliced ham using 100 ppm nisin. Beef jerky and sliced ham were treated with APP for 5 min and 9 min, respectively. In the bacterial solution, 100 ppm nisin out of 0-100 ppm nisin exhibited the highest bactericidal activity against L. monocytogenes (gram-positive bacteria; p<0.05); however, it did not exhibit bactericidal effects against E. coli O157:H7 (gram-negative bacteria). The APP+Nisin exhibited a 100% reduction rate in both E. coli O157:H7 and L. monocytogenes compared to the control group, and was more effective than the Nisin. The APP+Nisin decreased the number of colonies formed by 0.80 and 1.96 Log CFU/g for beef jerky and sliced ham, respectively, compared to the control, and exhibited a higher bactericidal effect compared to the Nisin (p<0.05). These results demonstrate the synergistic bactericidal effect of APP and nisin, providing a possible method to improve the limitations of nisin against gram-negative bacteria. In addition, this technology has the potential to be applied to various meats and meat products to control surface microorganisms.

Effect of plasma-activated organic acids on different chicken cuts inoculated with Salmonella Typhimurium and Campylobacter jejuni and their antioxidant activity.

Lactic acid, gallic acid, and their mixture (1% each) were prepared (LA, GA, and LGA) and plasma-activated organic acids (PAOA) were produced through exposure to plasma for 1 h (PAL, PAG, and PLGA). Chicken breast and drumstick were immersed in the prepared solutions for 10 min and analyzed their antibacterial effect against Salmonella Typhimurium and Campylobacter jejuni and antioxidant activity during 12 d of storage. As a result, PAOA inactivated approximately 6.37 log CFU/mL against S. Typhimurium and 2.76, 1.86, and 3.04 log CFU/mL against C. jejuni (PAL, PAG, and PLGA, respectively). Moreover, PAOA had bactericidal effect in both chicken parts inoculated with pathogens, with PAL and PLGA displaying higher antibacterial activity compared to PAG. Meanwhile, PAOA inhibited lipid oxidation in chicken meats, and PAG and PLGA had higher oxidative stability during storage compared to PAL. This can be attributed to the superior antioxidant properties of GA and LGA, including higher total phenolic contents, ABTS+ reducing activity, and DPPH radical scavenging activity, when compared to LA. In particular, when combined with plasma treatment, LGA showed the greatest improvement in antioxidant activity compared to other organic acids. In summary, PLGA not only had a synergistic bactericidal effect against pathogens on chicken, but also improved oxidative stability during storage. Therefore, PLGA can be an effective method for controlling microorganisms without adverse effect on lipid oxidation for different chicken cuts.

Identification and Characterization of Major Bile Acid 7α-Dehydroxylating Bacteria in the Human Gut.

The metabolism of bile acids (BAs) by gut bacteria plays an important role in human health. This study identified and characterized 7α-dehydroxylating bacteria, which are majorly responsible for converting primary BAs to secondary BAs, in the human gut and investigated their association with human disease. Six 7α-dehydratase (BaiE) clusters were identified from human gut metagenomes through sequence similarity network and genome neighborhood network analyses. Abundance analyses of gut metagenomes and metatranscriptomes identified a cluster of bacteria (cluster 1) harboring baiE genes that may be key 7α-dehydroxylating bacteria in the human gut. The baiE gene abundance of cluster 1 was significantly and positively correlated with the ratio of secondary BAs to primary BAs. Furthermore, the baiE gene abundances of cluster 1 were significantly negatively correlated with inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as advanced nonalcoholic fatty liver disease, liver cirrhosis, and ankylosing spondylitis. Phylogenetic and metagenome-assembled genome analyses showed that the 7α-dehydroxylating bacterial clade of cluster 1 was affiliated with the family Oscillospiraceae and may demonstrate efficient BA dehydroxylation ability by harboring both a complete bai operon, for proteins which produce secondary BAs from primary BAs, and a gene for bile salt hydrolase, which deconjugates BAs, in the human gut. IMPORTANCE In this study, we identified a key 7α-dehydroxylating bacterial group predicted to be largely responsible for converting primary bile acids (BAs) to secondary BAs in the human gut through sequence similarity network, genome neighborhood network, and gene abundance analyses using human gut metagenomes. The key bacterial group was phylogenetically quite different from known 7α-dehydroxylating bacteria, and their abundance was highly correlated with the occurrence of diverse diseases associated with bile acid 7α-dehydroxylation. In addition, we characterized the metabolic features of the key bacterial group using their metagenome-assembled genomes. This approach is useful to identify and characterize key gut bacteria highly associated with human health and diseases.

Metabolic Features of Ganjang (a Korean Traditional Soy Sauce) Fermentation Revealed by Genome-Centered Metatranscriptomics.

The taste and quality of soy sauce, a fermented liquid condiment popular worldwide, is greatly influenced by microbial metabolism during fermentation. To investigate the fermentative features of ganjang (a Korean traditional soy sauce), ganjang batches using meju (fermented soybean) bricks and solar salts were prepared, and organic compounds, microbial communities, metagenomes, and metatranscriptomes of ganjang were quantitively analyzed during fermentation. Polymeric compound analysis in the ganjang treated with/without microbial inhibitors revealed that indigenous enzymes of meju bricks might be primarily responsible for degrading polymeric compounds. Through metagenome binning and microbe sequencing, 17 high-quality genome sequences representing all major ganjang microbiota were obtained, and their transcriptional expressions were quantitatively analyzed by mapping metatranscriptome reads normalized by spike-in RNA sequencing to the 17 genomes, which revealed that microbial metabolism might primarily occur while meju bricks are in the ganjang solution and decrease significantly after the removal of meju bricks. Metabolic pathways for carbohydrates, proteins, and lipids of the major ganjang microbiota were reconstructed, and their metabolic genes were transcriptionally analyzed, revealing that facultative lactic acid fermentation by Tetragenococcus was the major fermentation process active in the ganjang fermentation and that aerobic respiration by facultatively aerobic bacteria such as Chromohalobacter, Halomonas, and Marinobacter was also an important metabolic process during fermentation. Although the abundances of Fungi and the corresponding transcriptional expression levels were generally much lower than those of Bacteria, our analysis suggests that yeasts such as Debaryomyces and Wickerhamomyces might be in large part responsible for producing biogenic amines and flavors. IMPORTANCE The taste and quality of soy sauce, a popular fermented liquid condiment worldwide, is greatly influenced by microbial metabolism during fermentation. Spontaneous fermentation of ganjang (a Korean traditional soy sauce) in a nonsterile environment leads to the growth of diverse bacteria and fungi during fermentation, making it difficult to understand the mechanism of ganjang fermentation. Genome-centered metatranscriptomic analysis, combined with organic compound analysis, quantitative metagenome and metatranscriptome analyses, and metabolic pathway reconstruction and expressional analysis of the major ganjang microbiota during fermentation, would provide comprehensive insights into the metabolic features of ganjang fermentation.

Metagenomic analysis of the human microbiome reveals the association between the abundance of gut bile salt hydrolases and host health.

Bile acid metabolism by the gut microbiome exerts both beneficial and harmful effects on host health. Microbial bile salt hydrolases (BSHs), which initiate bile acid metabolism, exhibit both positive and negative effects on host physiology. In this study, 5,790 BSH homologs were collected and classified into seven clusters based on a sequence similarity network. Next, the abundance and distribution of BSH in 380 metagenomes from healthy participants were analyzed. It was observed that different clusters occupied diverse ecological niches in the human microbiome and that the clusters with signal peptides were relatively abundant in the gut. Then, the association between BSH clusters and 12 human diseases was analyzed by comparing the abundances of BSH genes in patients (n = 1,605) and healthy controls (n = 1,540). The analysis identified a significant association between BSH gene abundance and 10 human diseases, including gastrointestinal diseases, obesity, type 2 diabetes, liver diseases, cardiovascular diseases, and neurological diseases. The associations were further validated by separate cohorts with inflammatory bowel diseases and colorectal cancer. These large-scale studies of enzyme sequences combined with metagenomic data provide a reproducible assessment of the association between gut BSHs and human diseases. This information can contribute to future diagnostic and therapeutic applications of BSH-active bacteria for improving human health.

Identification of a novel plant RNA virus species of the genus Amalgavirus in the family Amalgaviridae from chia (Salvia hispanica).

BACKGROUND: Chia (Salvia hispanica) is a flowering plant in the family Lamiaceae, which produces seeds that are a rich source of various nutritional compounds. OBJECTIVE: To identify a novel RNA virus potentially associated with chia. METHODS: Transcriptome data obtained from developing chia seeds were assembled into contigs. Sequence contigs containing an open reading frame (ORF) that showed amino acid identities with a viral RNA-dependent RNA polymerase (RdRp) were identified and analyzed. RESULTS: A genomic sequence of a novel plant RNA virus named Salvia hispanica RNA virus 1 (ShRV1) was identified in a chia seed transcriptome dataset. The ShRV1 genome sequence has two ORFs that showed high sequence identities with ORFs of known members of the genus Amalgavirus in the family Amalgaviridae. Amalgaviridae is a family of positive-sense double-stranded non-segmented RNA viruses that infect plants, fungi, and animals. The ShRV1 genome encodes two proteins: a putative replication factory matrix-like protein from ORF1 and an RdRp from the fused ORF of ORF1 and ORF2 by a + 1 programmed ribosomal frameshifting (PRF) mechanism. A conserved + 1 PRF motif sequence UUU_CGU was found at the ORF1/ORF2 boundary. A comparison of 31 amalgavirus ORF1 + 2 fusion proteins revealed that only three positions were repeatedly used as a + 1 PRF site during amalgavirus evolution. CONCLUSION: ShRV1 is a novel virus found to be associated with chia and may be useful for studying the molecular features of amalgaviruses.

Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.

Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data.

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites.

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

Loss of conserved ubiquitylation sites in conserved proteins during human evolution.

Ubiquitylation of lysine residues in proteins serves a pivotal role in the efficient removal of misfolded or unused proteins and in the control of various regulatory pathways by monitoring protein activity that may lead to protein degradation. The loss of ubiquitylated lysines may affect the ubiquitin­mediated regulatory network and result in the emergence of novel phenotypes. The present study analyzed mouse ubiquitylation data and orthologous proteins from 62 mammals to identify 193 conserved ubiquitylation sites from 169 proteins that were lost in the Euarchonta lineage leading to humans. A total of 8 proteins, including betaine homocysteine S­methyltransferase, clin and CBS domain divalent metal cation transport mediator 3, ribosome­binding protein 1 and solute carrier family 37 member 4, lost 1 conserved lysine residue, which was ubiquitylated in the mouse ortholog, following the human­chimpanzee divergence. A total of 17 of the lost ubiquitylated lysines are also known to be modified by acetylation and/or succinylation in mice. In 8 cases, a novel lysine evolved at positions flanking the lost conserved lysine residues, potentially as a method of compensation. We hypothesize that the loss of ubiquitylation sites during evolution may lead to the development of advantageous phenotypes, which are then fixed by selection. The ancestral ubiquitylation sites identified in the present study may be a useful resource for investigating the association between loss of ubiquitylation sites and the emergence of novel phenotypes during evolution towards modern humans.

Genome Sequences of Spinach Deltapartitivirus 1, Spinach Amalgavirus 1, and Spinach Latent Virus Identified in Spinach Transcriptome.

Complete genome sequences of three new plant RNA viruses, Spinach deltapartitivirus 1 (SpDPV1), Spinach amalgavirus 1 (SpAV1), and Spinach latent virus (SpLV), were identified from a spinach (Spinacia oleracea) transcriptome dataset. The RNA-dependent RNA polymerases (RdRps) of SpDPV1, SpAV1, and SpLV showed 72%, 53%, and 93% amino acid sequence identities with the homologous RdRp of the most closely related virus, respectively, suggesting that SpDPV1 and SpAV1 were novel viruses. Sequence similarity and phylogenetic analyses revealed that SpDPV1 belonged to the genus Deltapartitivirus of the family Partitiviridae, SpAV1 to the genus Amalgavirus of the family Amalgaviridae, and SpLV to the genus Ilarvirus of the family Bromoviridae. Based on the demarcation criteria, SpDPV1 and SpAV1 are considered as novel species of the genera Deltapartitivirus and Amalgavirus, respectively. This is the first report of these two viruses from spinach.

Genome Sequence of Spinach Cryptic Virus 1, a New Member of the Genus Alphapartitivirus (Family Partitiviridae), Identified in Spinach.

A distinct double-stranded RNA (dsRNA) cryptic virus, named spinach cryptic virus 1 (SpCV1), was identified from spinach transcriptome datasets. The SpCV1 genome has two dsRNA genome segments. The larger dsRNA1 has an open reading frame for a conserved RNA-dependent RNA polymerase (RdRp). The smaller dsRNA2 encodes a putative coat protein (CP). The sequence identity of SpCV1 RdRp and CP to the closest cryptic virus is 81% and 60%, respectively. Phylogenetic analysis indicates that SpCV1 is a novel member of the genus Alphapartitivirus (family Partitiviridae).