Characterization of a novel cold-adapted GH1 ß-glucosidase from Psychrobacillus glaciei and its application in the hydrolysis of soybean isoflavone glycosides.

The novel ß-glucosidase gene (pgbgl1) of glycoside hydrolase (GH) family 1 from the psychrotrophic bacterium Psychrobacillus glaciei sp. PB01 was successfully expressed in Escherichia coli BL21 (DE3). The deduced PgBgl1 contained 447 amino acid residues with a calculated molecular mass of 51.4 kDa. PgBgl1 showed its maximum activity at pH 7.0 and 40 °C, and still retained over 10% activity at 0 °C, suggesting that the recombinant PgBgl1 is a cold-adapted enzyme. The substrate specificity, Km, Vmax, and Kcat/Km for the p-Nitrophenyl-ß-D-glucopyranoside (pNPG) as the substrate were 1063.89 U/mg, 0.36 mM, 1208.31 U/mg and 3871.92/s, respectively. Furthermore, PgBgl1 demonstrated remarkable stimulation of monosaccharides such as glucose, xylose, and galactose, as well as NaCl. PgBgl1 also demonstrated a high capacity to convert the primary soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their respective aglycones. Overall, PgBgl1 exhibited high catalytic activity towards aryl glycosides, suggesting promising application prospects in the food, animal feed, and pharmaceutical industries.

Antigenic drift and epidemiological severity of seasonal influenza in Canada.

Seasonal influenza epidemics circulate globally every year with varying levels of severity. One of the major drivers of this seasonal variation is thought to be the antigenic drift of influenza viruses, resulting from the accumulation of mutations in viral surface proteins. In this study, we aimed to investigate the association between the genetic drift of seasonal influenza viruses (A/H1N1, A/H3N2 and B) and the epidemiological severity of seasonal epidemics within a Canadian context. We obtained hemagglutinin protein sequences collected in Canada between the 2006/2007 and 2019/2020 flu seasons from GISAID and calculated Hamming distances in a sequence-based approach to estimating inter-seasonal antigenic differences. We also gathered epidemiological data on cases, hospitalizations and deaths from national surveillance systems and other official sources, as well as vaccine effectiveness estimates to address potential effect modification. These aggregate measures of disease severity were integrated into a single seasonal severity index. We performed linear regressions of our severity index with respect to the inter-seasonal antigenic distances, controlling for vaccine effectiveness. We did not find any evidence of a statistical relationship between antigenic distance and seasonal influenza severity in Canada. Future studies may need to account for additional factors, such as co-circulation of other respiratory pathogens, population imprinting, cohort effects and environmental parameters, which may drive seasonal influenza severity.

Proteomic analysis of the Heliothis virescens ascovirus 3i (HvAV-3i) virion.

Ascoviruses are enveloped, circular, double-stranded DNA viruses that can effectively control the appetite of lepidopteran larvae, thereby reducing the consequent damage and economic losses to crops. In this study, the virion of a sequenced Heliothis virescens ascovirus 3i (HvAV-3i) strain was used to perform proteomic analysis using both in-gel and in-solution digestion. A total of 81 viral proteins, of which 67 were associated with the virions, were identified in the proteome of HvAV-3i virions. Among these proteins, 23 with annotated functions were associated with DNA/RNA metabolism/transcription, virion assembly, sugar and lipid metabolism, signalling, cellular homoeostasis and cell lysis. Twenty-one viral membrane proteins were also identified. Some of the minor 'virion' proteins identified may be non-virion contaminants of viral proteins synthesized during replication, identified by more recent and highly sensitive methods. The extensive identification of the ascoviral proteome will establish a foundation for further investigation of ascoviral replication and infection.

Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650 bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.

A rapid method for titration of ascovirus infectivity.

Ascoviruses are a recently described family and the traditional plaque assay and end-point PCR assay have been used for their titration. However, these two methods are time-consuming and inaccurate to titrate ascoviruses. In the present study, a quick method for the determination of the titer of ascovirus stocks was developed based on ascovirus-induced apoptosis in infected insect cells. Briefly, cells infected with serial dilutions of virus (10-2-10-10) for 24 h were stained with trypan blue. The stained cells were counted, and the percentage of nonviable cells was calculated. The stained cell rate was compared between virus-infected and control cells. The minimum-dilution group that had a significant difference compared with control and the maximum-dilution group that had no significant difference were selected and then compared each well of the two groups with the average stained cell rate of control. The well was marked as positive well if the stained cell rate was higher than the average stained cell rate of control wells; otherwise, the well was marked as negative wells. The percentage of positive wells were calculated according to the number of positive. Subsequently, the virus titer was calculated through the method of Reed and Muench. This novel method is rapid, simple, reproducible, accurate, and less material-consuming and eliminates the subjectivity of the other procedures for titrating ascoviruses.

The complete mitochondrial genome of Paragabara curvicornuta (Lepidoptera: Noctuoidea: Eribidae).

In this study, the complete mitochondrial genome (mitogenome) of Paragabara curvicornuta was sequenced for the first time by traditional PCR amplification and primer walking methods. As a circular DNA molecule, the entire mitogenome is 15,532 bp in length (GeneBank accession no. KT362742), and consists of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and an AT-rich region. The nucleotide composition is A (39.9%), C (12.0%), G (7.6%), and T (40.4%). Based onthe amino acid sequences of 13 PCGs from 43 noctuoid species as ingroups and two drepanoid species as outgroups, the phylogenetic trees was constructed. The phylogenetic relationships of the families among Noctuoidea are (Notodontidae, (Erebidae, (Nolidae, Noctuidae)). However, the phylogenetic position of P. curvicornuta is still unclear in the family Erebidae that is a monophyletic clade strongly supported by the bootstrap value of 100%.

Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of Heliothis virescens ascovirus 3 h (HvAV-3h).

Ascoviruses are double-stranded DNA viruses that mainly infect noctuid larvae, and are transmitted by the parasitoid wasp Microplitis similis Lyle. Ascovirus-parasitoids wasp-noctuid insects constitute the dissemination system. Selection of suitable reference genes for the dissemination system could play an important role in elucidating the pathogenic molecular mechanisms of ascovirus. Unfortunately, such studies on potential reference genes in the dissemination system of ascoviruses are lacking. In the present study, we evaluated 11 candidate reference genes: ß-actin1 (ACT1), ß-actin2 (ACT2), elongation factor 1 (EF1), elongation factor 2 (EF2), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), 28S ribosome (28S), Tubulin (TUB) and 18S ribosome (18S). The samples were originally from various virus concentrations and points-in-time of experimental treatments using RefFinder and four algorithms. The results showed that EF1 was the most stable internal gene in S. exigua and M. similis and that EF2 was the most stable in the IOZCAS-Spex-II-A cell line, and the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like (iap-like) genes from Heliothis virescens ascovirus 3 h (HvAV-3h). This study provides a crucial basis for future research that explores the molecular mechanisms of the pathogenesis of ascoviruses.

Genome sequence and organization analysis of Heliothis virescens ascovirus 3f isolated from a Helicoverpa zea larva.

The complete genome sequence of Heliothis virescens ascovirus 3f (HvAV-3f) was obtained. The HvAV-3f genome has a circular genome of 198,157bp with a G+C content of 46.0%, and encodes 190 open reading frames (ORFs) longer than 69 amino acids. Two major homologous regions (hrs) and 29 'baculovirus repeat ORFs' (bro) were found in the genome. BLAST analyses revealed that three HvAV-3f genes were homologous to that of lepidopteran insects. Nine ORFs were unique to HvAV-3f, in which two ORFs showed significant levels of similarity to genes that have not been previously described for ascoviruses in the Genbank database.