Amino Acid Sequence Divergence Research Articles

Restricted genetic heterogeneity of the Plasmodium vivax transmission-blocking vaccine (TBV) candidate Pvs48/45 in a low transmission setting: Implications for the Plasmodium vivax malaria vaccine development.

Plasmodium vivax is the most widespread malaria species parasitizing humans outside Africa, with approximately 100 million cases reported per year. Most human cases of P. vivax are asymptomatic with low parasitemia, making active case detection-based elimination programme challenging and less effective. Despite the widespread distribution of P. vivax, no effective vaccines are currently available. Transmission blocking vaccines have recently emerged as potential vaccine candidates to reduce transmission rates to below the essential levels required for the maintenance of the parasite life cycle. Here, we demonstrated that P. vivax was the predominant species found in a malaria-endemic area, although P. vivax/P. falciparum co-infections were also common. Through genomic sequence analysis and neighbor-joining algorithms, we demonstrated limited genetic heterogeneity in the P. vivax transmission-blocking vaccine candidate Pvs48/45 among clinical isolates of P. vivax. Restricted genetic polymorphism occurred at both nucleotide and amino acid levels. The most frequent mutation was A→G at nucleotide position 77 (46.7%), whereas the least frequent was C→T at nucleotide position 1230 (3.3%). The occurrence of single nucleotide polymorphisms (SNPs) distribution at 6/8 positions (75%) led to changes in amino acid sequences in the Pvs48/45 loci, whereas 2/8 (25%) of SNPs resulted in no amino acid sequence variations. Consistently, the nucleotide diversity in the Pvs48/45 locus among the P. vivax population studied was extremely low (π=0.000525). Changes in amino acid sequences in the Pvs48/45 protein did not result in substantial conformational modifications in the tertiary structures of these proteins. Unveiling the population genetic structure and genetic heterogeneity of vaccine target antigens are necessary for rational design of transmission-blocking antibody vaccines and to monitor the vaccine efficacy in clinical trials in endemic areas for malaria.

Sequence Analysis of the Complete Gene for Endoglucanase from Strains of Thermotolerant Bacillus

Three thermotolerant Bacillus isolates from the Philippines – identified as Bacillus subtilis (1280),Bacillus velezensis (1573), and Bacillus amyloliquefaciens (10105) – exhibited cellulase activity in carboxymethylcellulose (CMC) agar plate. Moreover, the expected size (approximately 1,500 bp) putative endoglucanase gene was amplified from all three local isolates. The endoglucanase gene amplified from a Bacillus licheniformis isolate (1320) was also used in the gene sequence analysis. A colorimetric dinitrosalicylic (DNS) acid assay revealed that the total endoglucanase activity of B. velezensis 1573 at 0.168 µM reducing sugar/min is significantly higher than the endoglucanase activity of the B. subtilis 1280 at 0.0673 µM rs/min and B. amyloliquefaciens 10105 at 0.0579 µM rs/min. Endoglucanase gene sequence analysis showed the highest pairwise identity between the endoglucanase gene of B. velezensis 1573 and B. amyloliquefaciens 10105 at 97.4%. The endoglucanase gene of B. licheniformis 1320 has the lowest pairwise identity at 74.6%, 76.2%, and 75.4% with B. amyloliquefaciens 10105, B. subtilis 1280, and B. velezensis 1573, respectively. Predicted amino acid sequences of the endoglucanases revealed variations at the signal peptide, catalytic core (CC) domain, linker, and carbohydrate-binding module (CBM). The endoglucanase of B. subtilis 1280 has a 506D insertion at the CBM domain. None of these variations, however, are located in regions implicated in catalytic reaction and substrate binding. Although the predicted protein structures using MODELLER revealed endoglucanases with good fit and high homology, variations in amino acid sequences may have resulted in the differences in enzyme activity and stability. The study generated gene and protein sequences as well as amplified genes that can be used as a starting template for enzyme improvement by rational design and site-specific mutagenesis or directed evolution using DNA shuffling.

Identification of QTLs Controlling Salt Tolerance in Cucumber (Cucumis sativus L.) Seedlings.

Cucumber is very sensitive to salt stress, and excessive salt content in soils seriously affects normal growth and development, posing a serious threat to commercial production. In this study, the recombinant inbred line (RIL) population (from a cross between the salt tolerant line CG104 and salt sensitive line CG37) was used to study the genetic mechanism of salt tolerance in cucumber seedlings. At the same time, the candidate genes within the mapping region were cloned and analyzed. The results showed that salt tolerance in cucumber seedlings is a quantitative trait controlled by multiple genes. In experiments carried out in April and July 2019, qST6.2 on chromosome six was repeatedly detected. It was delimited into a 1397.1 kb region, and nine genes related to salt tolerance were identified. Among these genes, Csa6G487740 and Csa6G489940 showed variations in amino acid sequence between lines CG104 and CG37. Subsequent qRT-PCR showed that the relative expression levels of both genes during salt treatment were significantly different between the two parents. These results provide a basis for the fine mapping of salt tolerant genes and further study of the molecular mechanism of salt tolerance in cucumber seedlings.

Acute Hepatitis C Virus Infections in Spouses: The Utility of a Genetic Analysis of the Hepatitis C Virus Hypervariable Region Sequence for Identifying the Infectious Source

A married couple who developed hepatitis was referred to our hospital. The husband was diagnosed with acute hepatitis C virus (HCV) infection based on HCV RNA positivity and seroconversion to HCV antibody. The wife was also diagnosed with HCV-related hepatitis; however, she could not be confirmed to have acute hepatitis due to the lack of information on her HCV negativity just before this event. The HCV strains recovered from the couple were genotype 2b and shared 100% identities within the 5’-untranslated region-core region sequence (655 nucleotides/nt) and non-structural (NS)5B region sequence (502 nt). The amplified hypervariable region 1 (HVR-1) sequence indicated that all 10 clones from the wife shared 100% identity and were identical to 3 of 10 heterogeneous clones (separable into 4 groups) from the husband. The husband had a history of intravenous drug use. These results suggested that one of four quasispecies 2b HCV strains was transmitted from the husband to the wife, with the husband being the infectious source for acute HCV infection in the wife, most likely via sexual intercourse. A sequence analysis of the HCV genomes and the further comparison of the HVR-1 amino acid sequence variability may be useful for defining the infectious source of HCV, especially in couples or cluster cases.

Bioinformatic analyses of kappa casein gene in mammalian livestock species

Kappa casein (CSN3) gene is a variant of the milk protein highly conserved in mammalian species. Genetic variations in CSN3 gene of six mammalian livestock species were investigated using bioinformatics approach. A total of twenty-seven CSN3 gene sequences with corresponding amino acids belonging to the six species studied were retrieved from GenBank. Using a comparative genomic approach, we obtained 1797 bp of the CSN3 sequences from cattle, goat, horse, pig, rabbit and sheep. Alignment of twenty-seven sequences within the region of 1713 bp and containing gaps was carried out using Clustal W. The sequence alignment revealed high polymorphisms of CSN3 sequences among the six species. The percent identity and similarity between species were determined by conducting pair-wise comparisons of the sequences. Results revealed high level of identity and similarity among the ruminant animals (84-97% and 86-99%) which implied the gene is conserved in all the ruminants. However, percent identity and similarity between pig and rabbit were relatively low (45%, 61%). This revealed that CSN3 protein from them might be functionally different and divergence between them is more ancient. Higher polymorphism of amino acids was observed among the species than within species. Four non-synonymous mutations occurred in cattle, six in goat, four in horse, three in rabbit and seven in sheep. The results of DnaSP analysis indicated that the selected region (1-1797 bp) of the twenty-seven sequences from all the species had 1298 nucleotide sites excluding sites with gaps (499). There are invariable and variable sites that include 185 singleton variable sites and 56 parsimony informative sites. The nucleotide diversity (ð= 0.212) and average number of nucleotide diversity (k = 94.84) for all sequences which were lower than the highest values in rabbit (ð = 0.557, k = 229) were estimated. Divergence of the species could be inferred from high genetic diversity of the CSN3 gene. The haplotype diversity was 1.00 in goat, horse and sheep indicating abundant genetic diversity in those species. Close relationship between goat and sheep was observed in phylogenetic tree of CSN3 gene which showed that the comparability of CSN3 gene sequences was highest between the goat and sheep and they evolved from a most recent common ancestor. A more detailed study involving more divergent taxa and complete amino acid sequence will help to delineate how the mutations affect the functions of the gene.

Computational Genomics of RELA gene of Pig and its protein prescription

RELA gene is a pleiotropic transcription that is present in almost all living cells where several signal transduction events end points were initiated by many arrays of stimuli that were related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis in the cells. The nucleotide and amino acid sequence (AAS) of RELA gene of pig and other mammalian species were downloaded from the National Center for Biotechnology information (NCBI) data base, United State of America and Universal protein resources (Uniprot) database, United Kingdom. Data generated were analyzed by bioinformatics tools. The results showed a high variation of AAS among the mammals where only four mammals have the same AAS length (551) with pig while others were not. The RELA gene of pig shared very high percent identity with all mammals ranging from 95.5% (warthog) to 83.5% (guinea pig) and similarity percentage ranged from 85.1 to 99.5% where the warthog had the highest percentage value while the least mammal was Guinea pig. The tree showed RELA gene with two clades; the first for mammals and the second for non-mammals. The RELA gene of pig was in the same cluster with the other Suidae family, warthog and babirusa. For the protein structure obtained, only 43% can be meaningfully predicted from the 61% residues modelled at more than 90%. The domain architecture of RELA protein structure contained mainly RNA Recognition motif and region of low complexity. The domainstarting position of all the organisms ranged from 191 to 210 while the termination of all the mammals ranged from 102 to 306 amino acids.The region of low complexity starting point ranged from position 173 to 462while termination positions ranged from 184 to 474. The evolution relationship of RELA gene of pig and other mammalian species were highly related and conserved among the mammalian species; they have high comparability and they evolved from common ancestors.

Characterization, expression patterns, and transcriptional responses of three core RNA interference pathway genes from Ostrinia nubilalis

RNA interference (RNAi) is commonly used in the laboratory to analyze gene function, and RNAi-based pest management strategies are now being employed. Unfortunately, RNAi is hindered by inefficient and highly-variable results when different insects are targeted, especially lepidopterans, such as the European corn borer (ECB), Ostrinia nubilalis (Lepidoptera: Crambidae). Previous efforts to achieve RNAi-mediated gene suppression in ECB revealed low RNAi efficiency with both double-stranded RNA (dsRNA) injection and ingestion. One mechanism that can affect RNAi efficiency in insects is the expression and function of core RNAi pathway genes, such as those encoding Argonaut 2 (Ago2), Dicer 2 (Dcr2), and a dsRNA binding protein (R2D2). To determine if deficiencies in these core RNAi pathway genes contribute to low RNAi efficiency in ECB, full-length complementary DNAs encoding OnAgo2, OnDcr2, and OnR2D2 were cloned, sequenced, and characterized. A comparison of domain architecture suggested that all three predicted proteins contained the necessary domains to function. However, a comparison of evolutionary distances revealed potentially important variations in the first RNase III domain of OnDcr2, the double-stranded RNA binding domains of OnR2D2, and both the PAZ and PIWI domains of OnAgo2, which may indicate functional differences in enzymatic activity between species. Expression analysis indicated that transcripts for all three genes were expressed in all developmental stages and tissues investigated. Interestingly, the introduction of non-target dsRNA into ECB second-instar larvae via microinjection did not affect OnAgo2, OnDcr2, or OnR2D2 expression. In contrast, ingestion of the same dsRNAs resulted in upregulation of OnDcr2 but downregulation of OnR2D2. The unexpected transcriptional responses of the core machinery and the divergence in amino-acid sequence between specific domains in each core RNAi protein may possibly contribute to low RNAi efficiency in ECB. Understanding the contributions of different RNAi pathway components is critical to adapting this technology for use in controlling lepidopteran pests that exhibit low RNAi efficiency.

Structural characterization of nonstructural protein 1 from SARS-CoV-2.

SummarySevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a single-stranded, enveloped RNA virus and the etiological agent of the current coronavirus disease 2019 pandemic. Efficient replication of the virus relies on the activity of nonstructural protein 1 (Nsp1), a major virulence factor shown to facilitate suppression of host gene expression through promotion of host mRNA degradation and interaction with the 40S ribosomal subunit. Here, we report the crystal structure of the globular domain of SARS-CoV-2 Nsp1, encompassing residues 13 to 127, at a resolution of 1.65 Å. Our structure features a six-stranded, capped β-barrel motif similar to Nsp1 from SARS-CoV and reveals how variations in amino acid sequence manifest as distinct structural features. Combining our high-resolution crystal structure with existing data on the C-terminus of Nsp1 from SARS-CoV-2, we propose a model of the full-length protein. Our results provide insight into the molecular structure of a major pathogenic determinant of SARS-CoV-2.

The Allelic Diversity of the Gibberellin Signaling Pathway Genes in Aegilops tauschii Coss.

Gibberellin-insensitive reduced height genes are widely spread in modern wheat varieties, making them resistant to lodging under conditions of intensive farming. However, the limited diversity of these genes present in wheat germplasm can limit the adaptability of newly created cultivars to the changing climate. The diversity of the gibberellin signaling pathway genes involved in plant height control—Reduced height 1 (Rht-D1), Gibberellin-insensitive dwarf 1 (Gid1-D) and Gibberellin-insensitive dwarf 2 (Gid2-D)—was studied in the diploid wild goatgrass Aegilops tauschii Coss., one of the ancestral species of the bread wheat (Triticum aestivum L.) and the donor of its D subgenome, using high-throughput sequencing. The examination of 24 Ae. tauschii accessions of different geographical origins revealed a large number of new alleles (haplotypes) not found in bread wheat varieties. Some of the detected polymorphisms lead to changes in the amino acid sequence of proteins. Four isoforms (amino acid sequence variants) were found for the RHT-D1 protein, and two isoforms—for the GID1 and GID2 proteins, each. An analysis of the co-occurrence frequencies of various isoforms of the three proteins showed that their combinations were not random in Ae. tauschii, which may indicate the functional significance of their differences. New alleles of the Rht-D1, Gid1-D, and Gid2-D genes are promising for introgression into bread wheat and studying their effect on plant height and adaptability.

Variations of Indole Metabolites and NRPS-PKS Loci in Two Different Virulent Strains of Xenorhabdus hominickii.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Variation in IgE binding potencies of seven Artemisia species depending on content of major allergens

BackgroundArtemisia weed pollen allergy is important in the northern hemisphere. While over 350 species of this genus have been recorded, there has been no full investigation into whether different species may affect the allergen diagnosis and treatment. This study aimed to evaluate the variations in amino acid sequences and the content of major allergens, and how these affect specific IgE binding capacity in representative Artemisia species.MethodsSix representative Artemisia species from China and Artemisia vulgaris from Europe were used to determine allergen amino acid sequences by transcriptome, gene sequencing and mass spectrometry of the purified allergen component proteins. Sandwich ELISAs were developed and applied for Art v 1, Art v 2 and Art v 3 allergen quantification in different species. Aqueous pollen extracts and purified allergen components were used to assess IgE binding by ELISA and ImmunoCAP with mugwort allergic patient serum pools and individual sera from five areas in China.ResultsThe Art v 1 and Art v 2 homologous allergen sequences in the seven Artemisia species were highly conserved. Art v 3 type allergens in A. annua and A. sieversiana were more divergent compared to A. argyi and A. vulgaris. The allergen content of Art v 1 group in the seven extracts ranged from 3.4% to 7.1%, that of Art v 2 from 1.0% to 3.6%, and Art v 3 from 0.3% to 10.5%. The highest IgE binding potency for most Chinese Artemisia allergy patients was with A. annua pollen extract, followed by A. vulgaris and A. argyi, with A. sieversiana significantly lower. Natural Art v 1-3 isoallergens from different species have almost equivalent IgE binding capacity in Artemisia allergic patients from China.Conclusion and clinical relevanceThere was high sequence similarity but different content of the three group allergens from different Artemisia species. Choice of Artemisia annua and A. argyi pollen source for diagnosis and immunotherapy is recommended in China.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

Whole Genome Sequence Analysis of Porcine Astroviruses Reveals Novel Genetically Diverse Strains Circulating in East African Smallholder Pig Farms.

Astroviruses (AstVs) are widely distributed and are associated with gastroenteritis in human and animals. The knowledge of the genetic diversity and epidemiology of AstVs in Africa is limited. This study aimed to characterize astroviruses in asymptomatic smallholder piglets in Kenya and Uganda. Twenty-four samples were randomly selected from a total of 446 piglets aged below 6 months that were initially collected for rotavirus study and sequenced for whole genome analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of seven strains with complete (or near complete) AstV genome revealed variable nucleotide and amino acid sequence identities with known porcine astrovirus (PoAstV) strains. The U083 and K321 strains had nucleotide sequence identities ranging from 66.4 to 75.4% with the known PoAstV2 strains; U460 strain had nucleotide sequence identities of 57.0 to 65.1% regarding the known PoAstV3; and K062, K366, K451, and K456 strains had nucleotide sequence identities of 63.5 to 80% with the known PoAstV4 strains. The low sequence identities (<90%) indicate that novel genotypes of PoAstVs are circulating in the study area. Recombination analysis using whole genomes revealed evidence of multiple recombination events in PoAstV4, suggesting that recombination might have contributed to the observed genetic diversity. Linear antigen epitope prediction and a comparative analysis of capsid protein of our field strains identified potential candidate epitopes that could help in the design of immuno-diagnostic tools and a subunit vaccine. These findings provide new insights into the molecular epidemiology of porcine astroviruses in East Africa.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 μM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Hemocyanin and hexamerinsexpression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Evolution of antigenic and genetic characteristics of foot-and-mouth disease virus serotype A circulating in Thailand, 2007–2019

Foot-and-mouth disease (FMD) is a persistent, major economic concern for livestock productivity, which is highly exacerbated by outbreaks in Thailand. FMD virus (FMDV) serotype A is more highly antigenic and genetically diverse than other serotypes, which has important implications for vaccine development as well as selection. Therefore, it is essential to continuously monitor antigenic and genetic changes of field isolates of FMDV serotype A. Here we used antisera against three vaccine strains (A/118/87, A/Sakolnakorn/97, and A/Lopburi/2012) to analyze the antigenicity of 133 field isolates of FMDV serotypes A in Thailand from 2007 to 2019. The majority of the isolates from 2007 to 2008 reacted only with the antiserum against strain A/118/87. In contrast, antigenic analysis revealed broad cross-reactivity and antigenic variations of the isolates from 2009 through 2019 against strains A/Sakolnakorn/97 and A/Lopburi/2012. These results indicate periodic changes in the antigenicity of field isolates of FMDV serotype A. Phylogenetic analysis of the VP1 region revealed that all isolates were of the Sea-97 lineage within the ASIA topotype. Analysis of the L-fragment genome sequences of 30 FMDV isolates collected throughout Thailand revealed highly variable amino acid sequences of VP1 and 3A, with the lowest average identity (94.56 %) and invariant (78.43 %) rates, respectively. The present findings indicate the importance of an active routine surveillance system incorporating antigenic and genetic analysis designated to continually update information about field isolates of FMDV serotype A. Such a system is essential for establishing and improving measures to control FMDV infections in Thailand and in neighboring Asian countries.

Phylogenetic and biological characterizations of a GI.3 norovirus.

Noroviruses (NoVs) are a major cause of acute non-bacterial gastroenteritis worldwide. In this study, we report the isolation, near-complete genome sequencing, and expression and biological characterization of the major capsid protein (VP1) of a GI.3 NoV isolated from a child presenting acute gastroenteritis. The genome of the GI.3 NoV is 7746bp in length, not including the poly-adenylation tail. Phylogenetic analysis based on the complete VP1 nucleotide sequences indicates that GI.3 NoVs could be divided into four clusters, with 4.6%, 5.3%, 6.6%, 1.9% intracluster variations in nucleotide and 4.8%, 3.8%, 6.1%, 1.7% intracluster variations in amino acid sequences, respectively. A Bayesian evolutionary analysis showed that GI.3 NoVs evolved at 2.44×10-3, 2.78×10-3, and 3.04×10-3 nucleotide substitutions/site/year using a strict clock model, an uncorrelated log-normal model (UCLN), and an uncorrelated exponential derivation model (UCED), respectively. VP1 protein expression using a recombinant baculovirus expression system leads to the successful assembly of virus-like particles (VLPs). In vitro VLP-Histo-blood group antigen (HBGA) binding assay indicates that GI.3 NoV VLPs strongly bind to blood type A salivary HBGAs, moderately bind to blood type O salivary HBGAs, and weakly bind or do not bind to blood type B and AB salivary HBGAs. In vitro VLP-HBGA binding blockade assay indicated that the binding of GI.3 NoV VLPs to blood type A salivary HBGAs could only be blocked by anti-GI.3 NoV VLPs serum but not non-GI.3 NoV genotype-specific hyperimmune sera (GI.2, GI.7, GII.4, GII.6, GII.7, and GII.17). The detailed characterization of GI.3 NoV in this study provides evidence that GI.3 NoV undergoes rapid evolution and exhibits no cross-blocking effects, suggesting that GI.3 NoV may potentially be utilized in the development of multivalent NoV vaccines.

Diversity of L1/L2 genes and molecular epidemiology of high-level carbapenem resistance Stenotrophomonas maltophilia isolates from animal production environment in China.

Stenotrophomonas maltophilia is emerging as a significant cause of human and animal disease worldwide. A total of 3400 samples were collected from animal farms and adjacent environments in China. The blaL1 and blaL2 genes were identified using whole genome sequence analyses and examined by phylogenetics. Isolates were also tested for susceptibility to 18 antibiotics. We isolated 118 strains of S. maltophilia from 3400 samples. The positive rates of blaL1 and blaL2 genes were 75% (89/118) and 22% (26/118) and we identified 11 L1 and 6 L2 amino acid sequence variants. S. maltophilia has at least two inducible β-lactamases (L1 and L2) that can hydrolyze almost all classes of β-lactams and these genes are suspected to confer carbapenem resistance. This represents a significant public health threat especially for hospitalized patients. We conducted a molecular surveillance study on the prevalence and characteristics of the blaL1 and blaL2 genes of S. maltophilia.

Functional divergence of principal alcohol o-acyltransferase for biosynthesis of volatile acetate esters among tomato wild species (Solanum Sect. Lycopersicon)

Volatile esters are the chemicals that have multiple physiological functions including plant defense responses and reproduction. From a human perspective, the esters largely contribute to the fruity aroma of freshy fruits. Composition of volatile esters show a significant diversity among the wild tomato species (Solanum sect. Lycopersicon). To address the basis for this divergence, here we conducted functional analysis of a gene encoding major alcohol o-acyltransferase (AAT1) that catalyzes volatile ester formation. Although AAT1 transcripts were highly expressed in the ripe fruits of all the wild species examined, their enzymatic properties significantly differed due to amino acid sequence variations. Notably, AAT1s from S. pennellii showed the highest ability to produce acetate esters whereas AAT1s from S. neorickii, S. chmielewskii and S. habrochaites had the lowest activities. Further, screenings using domain-swapped or point-mutated AAT1s allowed us to identify Met/Thr352 as one of the critical residues related to the transferase activity with acetyl-CoA. This finding is potentially applied to aroma engineering in which a site-directed mutagenesis at this position in alcohol o-acyltransferases could enable to manipulate volatile ester levels in ripe fruits.

Genomic Characteristics and Potential Metabolic Adaptations of Hadal Trench Roseobacter and Alteromonas Bacteria Based on Single-Cell Genomics Analyses.

Heterotrophic bacteria such as those from the Roseobacter group and genus Alteromonas dominate the hadal zones of oceans; however, we know little about the genomic characteristics and potential metabolic adaptations of hadal trench-dwelling bacteria. Here, we report multiple single amplified genomes (SAGs) belonging to Roseobacter and Alteromonas, recovered from the hadal zone of the Mariana Trench. While phylogenetic analyses show that these hadal SAGs cluster with their surface relatives, an analysis of genomic recruitment indicates that they have higher relative abundances in the hadal zone of the Mariana Trench. Comparative genomic analyses between the hadal SAGs and reference genomes of closely related shallow-water relatives indicate that genes involved in the mobilome (prophages and transposons) are overrepresented among the unique genes of the hadal Roseobacter and Alteromonas SAGs; the functional proteins encoded by this category of genes also shows higher amino acid sequence variation than those encoded by other gene sets within the Roseobacter SAGs. We also found that genes involved in cell wall/membrane/envelope biogenesis, transcriptional regulation, and metal transport may be important for the adaptation of hadal Roseobacter and Alteromonas lineages. These results imply that the modification of cell surface-related proteins and transporters is the major direction of genomic evolution in Roseobacter and Alteromonas bacteria adapting to the hadal environment, and that prophages and transposons may be the key factors driving this process.