Streamlined Whole Genome Sequencing of Mumps for High Resolution Outbreak Analysis

Since 2015, the United States has experienced a resurgence in the number of mumps cases and outbreaks in fully vaccinated populations. These outbreaks have occurred predominantly in close quarter settings such as camps, colleges and detention centers. Phylogenetic analysis of 758 mumps positive samples from outbreaks across the United States, identified 743 (98%) as genotype G based on sequence analysis of the mumps small hydrophobic (SH) gene. Additionally, SH sequences in the genotype G samples showed almost no sequence diversity, with 675 (91%) of them having identical sequences or only one nucleotide difference. This uniformity of circulating genotype and strain created complications for epidemiologic investigations and necessitated the development of a system for rapidly generating mumps whole genome sequences for more detailed analysis. In this study, we report a novel and streamlined assay for whole genome sequencing (WGS) of mumps virus genotype G. The WGS procedure successfully generated 318 high-quality WGS sequences on nucleic acid from genotype G-positive respiratory samples collected during several mumps outbreaks in the United States between 2016-2019. Sequencing was performed by a rapid and highly sensitive custom Ion AmpliSeq mumps genotype G panel, with sample preparation performed on an Ion Chef and sequencing on an Ion S5. The WGS data generated by the AmpliSeq panel provided enhanced genomic resolution for epidemiological outbreak investigations. Translation and protein sequence analysis also identified several potentially important epitope changes in the circulating mumps genotype G strains compared to the Jeryl-Lynn strain (JL5) used in vaccines in the United States which could explain the current level of vaccine escapes.

BioSeq-BLM: a platform for analyzing DNA, RNA and protein sequences based on biological language models

In order to uncover the meanings of ‘book of life’, 155 different biological language models (BLMs) for DNA, RNA and protein sequence analysis are discussed in this study, which are able to extract the linguistic properties of ‘book of life’. We also extend the BLMs into a system called BioSeq-BLM for automatically representing and analyzing the sequence data. Experimental results show that the predictors generated by BioSeq-BLM achieve comparable or even obviously better performance than the exiting state-of-the-art predictors published in literatures, indicating that BioSeq-BLM will provide new approaches for biological sequence analysis based on natural language processing technologies, and contribute to the development of this very important field. In order to help the readers to use BioSeq-BLM for their own experiments, the corresponding web server and stand-alone package are established and released, which can be freely accessed at http://bliulab.net/BioSeq-BLM/.

Emergence of a tet(M) Variant Conferring Resistance to Tigecycline in Streptococcus suis

The aim of this study was to gain insight into the resistance determinants conferring resistance to tigecycline in Streptococcus (S.) suis and to investigate the genetic elements involved in their horizontal transfer. A total of 31 tetracycline-resistant S. suis isolates were screened for tigecycline resistance by broth microdilution. S. suis isolate SC128 was subjected to whole genome sequencing with particular reference to resistance determinants involved in tigecycline resistance. Transferability of genomic island (GI) GISsuSC128 was investigated by transformation. The roles of tet(L) or tet(M) in contributing to tigecycline resistance in S. suis were confirmed by transformation using different tet(L)- or tet(M)-carrying constructs. Only S. suis SC128 showed a tigecycline resistance phenotype. A tet(L)-tet(M) and catA8 co-carrying GISsuSC128 was identified in this isolate. After transfer of the novel GI into a susceptible recipient, this recipient showed the same tigecycline resistance phenotype. Further transfer experiments with specific tet(L)- or tet(M)-carrying constructs confirmed that only tet(M), but not tet(L), contributes to resistance to tigecycline. Protein sequence analysis identified a Tet(M) variant, which is responsible for tigecycline resistance in S. suis SC128. It displayed 94.8% amino acid identity with the reference Tet(M) of Enterococcus faecium DO plasmid 1. To the best of our knowledge, this is the first time that a tet(M) variant conferring resistance to tigecycline was identified in S. suis. Its location on a GI will accelerate its transmission among the S. suis population.

Fine mapping of the BnaC04.BIL1 gene controlling plant height in Brassica napus L

Background Plant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2. Results The inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2, 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2, which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture. Conclusions A dominant dwarfing gene, BnaC04.BIL1, encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.

Evolutionary Analysis of Transcriptional Regulation Mediated by Cdx2 in Rodents

Background: Differences in gene expression, which arises from divergence in cis-regulatory elements or alterations in transcription factors (TFs) binding specificity, are one of the most important causes of phenotypic diversity during evolution. On one hand, changes in the cis-elements located in the vicinity of target genes affect TF binding and/or local chromatin environment, thereby modulating gene expression in one-to-one manner. On the other hand, alterations in trans-factors influence the expression of their target genes in a more pleiotropic fashion. Although evolution of amino acid sequences is much slower than that of non-coding regulatory elements, particularly for the TF DNA binding domains (DBD)， it is still possible that changes in TF-DBD might have the potential to drive large phenotypic changes if the resulting effects have a net positive effect on the organism’s fitness. If so, species-specific changes in TF-DBD might be positively selected. So far, however, this possibility has been largely unexplored.Results: By protein sequence analysis, we observed high sequence conservation in the DNA binding domain (DBD) of the transcription factor Cdx2 across many vertebrates, whereas three amino acid changes were exclusively found in mouse Cdx2 (mCdx2), suggesting potential positive selection in the mouse lineage. Multi-omics analyses were then carried out to investigate the effects of these changes. Surprisingly, there were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Finally, we used rat-mouse allodiploid embryonic stem cells (RMES) to study the cis effects of Cdx2-mediated gene regulation between the two rodents. Interestingly, whereas Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.Conclusions: There were no significant functional differences between mCdx2 and its rat homologue (rCdx2), and none of the three amino acid changes had any impact on its function. Moreover, Cdx2 binding is largely divergent between mouse and rat, the transcriptional effect induced by Cdx2 is conserved to a much larger extent.

Molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014 to 2019

The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses.

Comparative Analyses of ACE2 Receptor Binding Corona Viruses that Cause Mild versus Severe Acute Respiratory Syndrome in Humans

Aim: To analyze the spike proteins and Replication-Transcription Complexes (RTCs) of the Mild and Severe Acute Respiratory Syndrome (SARS) and SARS-related coronaviruses (CoVs) to find out the similarities and differences between them, as both of groups bind to angiotensin-converting enzyme 2 (ACE2) receptor for human cell entry. Study Design: Bioinformatics, Biochemical, Site-Directed Mutagenesis (SDM), X-ray crystallographic, cryo-Electron microscopic (cryo-EM) and Mass Spectrometric (MS) data were analyzed. Methodology: The protein sequence data for spike proteins and the proteins of the RTCs, viz. the RNA- dependent RNA polymerases (RdRps), primases and the nonstructural protein 7 (NSP7) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic and cryo-EM studies on these proteins were used to confirm the possible amino acids involved in ACE2 receptor binding and active sites of the RTCs. For identification of probable N-linked and O-linked glycosylation sites, NetNGlyc 1.0 and NetOGlyc 4.0 tools of Technical University of Denmark were used. ExPASy tool was used for pI analysis. Results: The spike protein of human CoV (HCoV)-NL63 is ~90 amino acids longer than the spike proteins of SARS and SARS-related CoVs. The additions are mostly found in the N-terminal regions and few insertions are also found in the crucial receptor binding domain (RBD). The SARS and SARS-related CoVs and HCoV-NL63 showed several conserved residues, motifs and large peptide regions. The most important aspect between the recent pandemic causing SARS-CoV-2 and HCoV-NL63 is a unique but different tetrapeptide insertions very close to the S1/S2 cleavage region, i.e., -PRRA- and -IPVR-, respectively. The next cleavage point S2’ and the transmembrane domains are conserved between the two groups. The RdRps are highly conserved between the two groups. The catalytic regions, catalytic amino acids and the NTP selection tripeptide regions are completely conserved between SARS-CoVs and HCoV-NL63. However, one of the metal binding sites, viz. the universal –GDD- reported in all RdRps is aligning with– KDG- in the RdRp of HCoV-NL63. The other metal binding site, viz. –SDD- is completely conserved in both the groups. The NiRAN domains of the RdRps differed from the possible catalytic amino acid and NTP selection tripeptide regions. The primases (NSP8) and the NSP7 subunits of the RTC are highly conserved in both the groups. The NSP8 and NSP7 subunits exhibit closer similarities between the MERS-CoV and HCoV-NL63. Unlike other SARS and SARS-related CoVs, the HCoV-NL63 possesses only a single accessory protein. Interestingly, a large number of amino acids are replaced with Ns in the spike proteins (which is also reflected in the number of N-linked glycosylation sites in it) as well as in the RTC. Conclusions: Detailed analysis revealed several unique features in the HCoV-NL63 pathogen. As all the pandemic strains like SARS-CoV-1, SARS-CoV-2 and the milder HCoV-NL63 strain, use the same ACE2 receptor for entry into human cells, the frequent infection of humans by HCoV-NL63, especially in children, suggests that there is an ample opportunity for highly pathogenic variants to evolve in the future.

Comprehensive in silico structural-functional analysis of Enterobacter GH19 class I chitinase (chiRAM) gene: cloning and heterologous expression

I. Background. Present study aims to clone and express the gene-encoding chitinase / GH19 family from Enterobacter sp. in E.coli with in silico sequence analyses.. II. Methods and results. The putative open reading frame of 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 chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). chiRAM in silico protein sequence analysis revealed GH19 class I chitinase: N-terminus signal peptide (Met1-Ala23), catalytic domain (Val83-Glu347 & catalytic triad Glu149, Glu171, Ser218), proline-rich hinge (Pro414 -Pro450), (polycystic kidney disease protein motif (Gly 465-Ser 533), C-terminus chitin-binding domain (Ala553- Glu593), and class I conserved motifs (NYNY and AQETGG). Three dimensional model was constructed by LOMETS MODELLER, PDB template: 2dkvA (Oryza sativa L. japonica class I chitinase). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72kDa; SDS-PAGE) in 1.0 mM IPTG induced E.coli BL21 (DE3) Rosetta at room temperature, 18 hrs post induction. Optimized expression yielded active chiRAM with 1.974 U/mL ± 0.0002, on shrimp colloidal chitin (SCC), in induced E.coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified the 72 kDa band in the soluble fraction with 52.3% coverage sequence exclusive to Enterobacter cloacae chitinase/GH19 (WP_063869339.1). III. Conclusions. Despite the successful cloning and expression of chiRAM of Enterobacter sp. in E.coli with an appreciable chitinase activity, prospective studies would focus on minimizing IBs to facilitate chiRAM purification and characterization.

Molecular Characterization of Ahp2, a Lytic Bacteriophage of Aeromonas hydrophila

Aeromonas hydrophila is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects A. hydrophila. We observed that 96% of the Ahp2 particles adsorbed to A. hydrophila within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of Aeromonas phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect Aeromonas hydrophila and Aeromonas salmonicida. The tail module encompassing ORF27-29 in the Ahp2 genome was present in all Aeromonas phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses A. hydrophila AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic Aeromonas phage and could be a candidate for therapeutic phage cocktails.