Isolation and Characterization of Bovine RVA from Northeast China, 2017–2020

Group A rotaviruses (RVAs) are major enteric pathogens causing infections in calves. To investigate the epidemiological characteristics and genetic diversity of bovine rotavirus (BRV), 233 fecal samples were collected from calves with diarrhea in northeast China. The samples were analyzed for sequences encoding the inner capsid protein VP6 (subgroup) and the outer capsid proteins VP7 and VP4 (G and P type, respectively) using RT-PCR. Ten of the 233 samples (4.3%) were identified as BRV positive and were used for virus isolation and sequence analysis, revealing that all strains analyzed were of the G6P[1] genotype. The isolates exhibited high VP6 sequence identity to the USA cow RVA NCDV strain (>99% amino acid identity) and were further shown to be closely related to Japanese cow RVA BRV101 and Israelian human RVA G6P[1] strains, with >99% amino acid identity to VP7 and VP4 proteins, respectively. Comparative analyses of genome-predicted amino acid sequences between the isolates and the NCDV strains indicated that the antigenicity and infectivity of the strains isolated had changed. In this study, BRV genotypes and the genetic diversity among vaccinated cattle herds were monitored to provide epidemiological data and references for early diagnosis, allowing for early detection of new, potentially pathogenic RVA strains.

Zooshikella harenae sp. nov., Isolated from Pacific Oyster Crassostrea gigas, and Establishment of Zooshikella ganghwensis subsp. marina subsp. nov. and Zooshikella ganghwensis subsp. ganghwensis subsp. nov.

Here, we describe the polyphasic taxonomy of a novel isolated strain WH53T from the genus Zooshikella isolated from the sand sediment located between the lumen of the Crassostrea gigas From Germany. Phylogenetic analysis determined that the strain WH53T had a high similarity to Zooshikella ganghwensis JC2044T (99.57%) and Zooshikella marina LMG 28823T (99.36%). Strain WH53T contained ubiquinone-9 (Q-9) as the predominant menaquinone, and the major fatty acids were C16:0, C16:1ω7c, and C18:1ω7c. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, amino phospholipids, and unidentified phospholipids were identified as their polar lipid composition. The DNA G+C content and genome size of strain WH53T were 40.08 mol% and 5,914,969 bp, respectively. Digital DNA–DNA hybridisation (dDDH) for strain WH53T against Z. ganghwensis JC2044T and Z. marina LMG 28823T showed low relatedness values of 26.3% and 26.1%, respectively. The extract of strain WH53T exhibited antimicrobial property. Strain WH53T represents a novel species in the genus Zooshikella. We propose the name of Zooshikella harenae sp. nov., with the type strain WH53T (= DSM 111628T = NCCB 100808T). Furthermore, the dDDH, average nucleotide identity (ANI), percentage of conserved proteins (POCP), and amino acid identity (AAI) value between Z. marina LGM 28823T and Z. ganghwensis DSM 15267T were 79.9%, 97.84%, 76.08%, and 87.01%, respectively, suggesting that both of them should be reclassified as Z. ganghwensis subsp. marina subsp. nov. and Z. ganghwensis subsp. ganghwensis DSM 15267 subsp. nov.

Discovery of UPSTREAM of FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) in Gladiolus ×Hybridus, G. Dalenii

Background. Gladiolus is a geophytic floricultural crop cultivated for cut flower and garden ornamental uses. Monocotyledonous flower crops have few, if any, flowering genes identified. Ornamental geophytes such as gladiolus, lily, tulip and daffodil are examples of floral crops that are currently being investigated to understand the flowering pathway. While the flower genes and environmental / hormonal factors leading to flowering are established in Arabidopsis, the lack of identified flowering genes in economically important ornamental geophytic crops, such as gladiolus, is critical to further genetic research. Thus, the importance of such an ornamental crop that relies on flowers (flowering) for economic purposes encourages researchers to discover the flowering genes to breed vigorous, flowering cultivars. The understanding of the flowering mechanisms in the flowering pathway is also of paramount importance. Results. Herein we show the discovery of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) genes in Gladiolus ×hybridus and G. dalenii. The UFC gene is adjacent to FLOWERING LOCUS C (FLC) which is a floral repressor in many temperate species. The FLX gene upregulates FRIGIDA (FRI) which upregulates FLC expression. Discovery of both genes is a step forward in finding the FLC gene in gladiolus, provided they are linked. Seventeen gladiolus genotypes, consisting of early flowering and commercial cultivars, were discovered to possess the UFC gene, consisting of four exons in two allelic forms. The sequenced UFC gene, when translated into its amino acid sequence and set in pair-alignment to other species, has up to 57% in amino acid identity to Musa acuminata. The UFC protein ranges in identity with pair-alignment to other monocot species, also with 57% amino acid identity to M. acuminata. The FLX gene in gladiolus has 3/5 (60%) exons in common relative to Ananas comosus, i.e. lacking 2 exons and a partially complete gene sequence; the pair-alignment of the three exons shows an overall ~65% identity of FLX to A. comosus. The UFC protein consists of a conserved domain, DUF966, which is higher in identity (86%) and pair-alignment with Elaeis guineensis. Conclusions. The two newly-discovered genes in gladiolus, UFC and FLX, provide insight to further our understanding of the flowering mechanism, flowering pathway genes, and vernalization response in ornamental geophytes. This knowledge will be valuable for gladiolus breeders and geneticists to finding the FLC gene, identify segregating seedlings for both UFC and FLX, and aid in marker assisted selection for flowering gene expression.

Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity

Olfactory receptor repertoires exhibit remarkable functional diversity, but how these proteins have evolved is poorly understood. Through analysis of extant and ancestrally-reconstructed drosophilid olfactory receptors from the Ionotropic receptor (Ir) family, we investigated evolution of two organic acid-sensing receptors, Ir75a and Ir75b. Despite their low amino acid identity, we identify a common 'hotspot' in their ligand-binding pocket that has a major effect on changing the specificity of both Irs, as well as at least two distinct functional transitions in Ir75a during evolution. Moreover, we show that odor specificity is refined by changes in additional, receptor-specific sites, including those outside the ligand-binding pocket. Our work reveals how a core, common determinant of ligand-tuning acts within epistatic and allosteric networks of substitutions to lead to functional evolution of olfactory receptors.

Molecular Epidemiological Study of Porcine Epidemic Diarrhea from Winter 2020 to Spring 2021 in Jiangsu Province

Background: Porcine epidemic diarrhea (PED) is an acute and highly contagious infectious disease caused by the porcine epidemic diarrhea virus (PEDV) that occurs most frequently from winter to spring. It is associated with high morbidity and mortality rates, especially among piglets, and causes huge losses in the pig industry. The aim of this molecular epidemiological study was to identify the current strains of PEDV that are prevalent in Jiangsu Province, China.Methods: From winter 2020 to spring 2021, 793 small intestine tissue, fecal, and anal swab samples were collected from 72 pig farms in 11 counties in the jurisdiction of 5 regions of Jiangsu Province (Yancheng, Suqian, Changzhou, Xuzhou, and Yangzhou). A highly variable region of the S gene was amplified and sequenced, and phylogenetic analysis was conducted to compare this sequence with corresponding sequences from reference strains deposited in GenBank. Results: A total of 457 samples from 57 pig farms were positive for PEDV: this implies a positivity rate of 79% (57/72) for pig farms and a sample positivity rate of 57.6% (457/793). The positivity rates were 78% (107/137) in Yancheng, 53% (218/409) in Suqian, 48% (94/195) in Changzhou, 80% (16/20) in Xuzhou, and 88% (14/16) in Yangzhou. Seven representative samples were selected for sequencing, and phylogenetic analysis showed that the seven isolated strains exhibited 88.0%–100% nucleotide identity and 87.3%–99% amino acid identity. Additionally, our isolates exhibited 88.3%–99.7% nucleotide identity and 88%–98.5% amino acid identity with the reference PEDV strains. Phylogenetic tree analysis indicated that there were considerable difference in the sources of the variants.Conclusions: PEDV had a high infection rate among pigs and is possibly the main pathogenic agent of pig diarrhea in Jiangsu province. Importantly, vaccines must be screened for their efficacy against the newly identified variants.

Identification of a novel metallo-β-lactamase, VAM-1, in a foodborne Vibrio alginolyticus isolate from China

A multidrug-resistant Vibrio alginolyticus isolate recovered from a shrimp sample with reduced carbapenem susceptibility produced a novel metallo-β-lactamase, VAM-1. That carbapenemase shared 67% to 70% amino acid identity with several VMB family subclass B1 MBLs which were recently reported among some marine bacteria including Vibrio , Glaciecola and Thalassomonas . The bla VAM-1 gene was located in a novel conjugative plasmid, namely pC1579 and multiple copies of bla VAM-1 via an unusual mechanism of gene amplification were detected in pC1579. These findings underline the emergence of marine organisms acting as natural reservoirs for MBL genes and the importance of continuous bacterial antibiotic resistance surveillance.

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.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

A 1958 Isolate of Kedougou Virus (KEDV) from Ndumu, South Africa, Expands the Geographic and Temporal Range of KEDV in Africa

The mosquito-borne flavivirus, Kedougou virus (KEDV), first isolated in Senegal in 1972, is genetically related to dengue, Zika (ZIKV) and Spondweni viruses (SPOV). Serological surveillance studies in Senegal and isolation of KEDV in the Central African Republic indicate occurrence of KEDV infections in humans, but to date, no disease has been reported. Here, we assembled the coding-complete genome of a 1958 isolate of KEDV from a pool of Aedes circumluteolus mosquitoes collected in Ndumu, KwaZulu-Natal, South Africa. The AR1071 Ndumu KEDV isolate bears 80.51% pairwise nucleotide identity and 93.34% amino acid identity with the prototype DakAar-D1470 strain and was co-isolated with SPOV through intracerebral inoculation of suckling mice and passage on VeroE6 cells. This historical isolate expands the known geographic and temporal range of this relatively unknown flavivirus, aiding future temporal phylogenetic calibration and diagnostic assay refinement.