Meng Qu

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses.

Listeria monocytogenes is a high-risk pathogenic bacterium associated with ready-to-eat foods and poses a potential threat to consumer health. This study aimed to investigate the prevalence, characterization and genetic diversity of L. monocytogenes in ready-to-eat raw salmon and trout products obtained from physical stores and online stores in China. Out of 150 samples analyzed, 23 (15.3%) were positive for L. monocytogenes. Among these positive samples, three (12%) were from Japanese restaurants, four (16%) from farmers markets, one (2.9%) from large supermarkets and fifteen (30%) from e-commerce platforms, and only one sample showed a contamination level exceeding 100 most probable number (MPN)/g. The isolates from positive samples demonstrated a concrete public health risk through several findings: twenty-three L. monocytogenes exhibited varying degrees of cytotoxicity, ranging from 7.6% to 71.8%. Compared with the reference strain ATCC 19115, five of these isolates were highly cytotoxic, a result that was validated by mouse survival rate experiment, which also confirmed their high virulence at tested dose. All isolates were resistant to cefuroxime sodium, ceftriaxone, cefepime and nalidixic acid, and 13% showed resistance to sulphamethoxazole-trimethoprim. Three serogroups were identified, with serogroup Ⅰ.1 (1/2a, 3a) being the most prevalent (65.2%). These isolates were grouped into eight sequence types, with ST8 (34.8%) and ST87 (30.4%) dominating. All isolates carried virulence genes associated with LIPI-1 andmultiple internalin genes (inlA, inlB, inlJ and inlK), confirming their potential pathogenicity. Additionally, the isolates harbored antimicrobial resistance genes lin and FosX. The five highly virulent isolates exhibited the highest genetic similarity to J2-031 (GCA_000438645.1) and C1-387 (GCA_000438605.1). The results provided valuable information for Chinese regulatory authorities to strengthen the risk monitoring of L. monocytogenes in ready-to-eat raw salmon and trout products.

Tuna are economically important as food resources in food markets. However, because tuna is often processed into steaks or fillets, the meat can be difficult to identify through morphological features. For effective fishery management and to protect the rights of consumers, it is necessary to develop a molecular method to accurately identify the species used in tuna products. Herein, we discovered five single-nucleotide polymorphism (SNP) sites via 2b-RAD sequencing and developed five SNP-based real-time polymerase chain reaction assays for the rapid identification of five highly priced tuna species. Three species-specific TaqMan systems were designed to identify albacore tuna (Thunnus alalunga), bigeye tuna (T. obesus), and southern bluefin tuna (T. maccoyii) and two cycling systems were designed to identify yellowfin tuna (T. albacares) and Atlantic bluefin tuna (T. thynnus). The systems showed good specificity and sensitivity (sensitivity of 0.0002 ng μL⁻¹ for albacore tuna, bigeye tuna, and southern bluefin tuna and 0.002 ng μL⁻¹ for yellowfin tuna and Atlantic bluefin tuna). Both systems were able to distinguish the target species from other species in a specific, sensitive, and accurate manner. Thus, these methods can be employed for the identification of species used in tuna products, protecting consumers and producers from economic fraud.

Porcine circovirus-associated diseases, caused by porcine circovirus type 2 (PCV2), are widespread and result in significant economic losses to the global swine industry. PCV2 can currently be divided into nine genotypes (PCV2a to PCV2i), with the currently dominant one being the PCV2d genotype. In this study, 2675 samples from 804 pig farms in 13 cities in Jiangsu Province, China, were collected between 2014 and 2021 and subjected to polymerase chain reaction analysis to investigate the frequency and genetic diversity of PCV2. The results showed that 41.42% (1108/2675) of samples tested positive for PCV2. The researchers further analyzed the genetic characteristics of 251 PCV2 strains and found that they belonged to the following four genotypes: PCV2a, PCV2b, PCV2d, and PCV2i. The dominant genotype was PCV2d, with a frequency of 49.80% (125/251). The detection rate of PCV2b was significantly higher than those of PCV2a and PCV2i, at 35.46% (89/251), 7.57% (19/251), and 7.17% (18/251), respectively. The percentage of different genotypes of PCV2 varied irregularly over time. We have further revealed the fingerprint of PCV2i genomic nucleotides for the first time. In conclusion, this study illustrates the high frequency and evolutionary features of PCV2 in Jiangsu Province over the past few years.

Histo-blood group antigens (HBGAs) comprise a family of cell-surface carbohydrates that are considered norovirus-specific binding receptors or ligands. HBGA-like molecules have also been detected in oysters as common norovirus carriers, although the pathway involved in the synthesis of these molecules in oysters has yet to be elucidated. We isolated and identified a key gene involved in the synthesis of HBGA-like molecules, FUT1, from Crassostrea gigas, named CgFUT1. Real-time quantitative polymerase chain reaction analysis showed that CgFUT1 mRNA was expressed in the mantle, gill, muscle, labellum, and hepatopancreatic tissues of C. gigas, with the hepatopancreas exhibiting the highest expression level. A recombinant CgFUT1 protein with a molecular mass of 38.0 kDa was expressed in Escherichia coli using a prokaryotic expression vector. A eukaryotic expression plasmid was constructed and transfected into Chinese hamster ovary (CHO) cells. The expression of CgFUT1 and membrane localization of type H-2 HBGA-like molecules in CHO cells were detected using Western blotting and cellular immunofluorescence, respectively. This study indicated that CgFUT1, expressed in C. gigas tissues, can synthesize type H-2 HBGA-like molecules. This finding provides a new perspective for analyzing the source and synthetic pathway of HBGA-like molecules in oysters.