Whole genome sequencing and genome characterization of Aichivirus isolated from Korean adults.

The whole-genome sequence (WGS) analysis of Aichivirus (AiV) identified in Korea was performed in this study. Using Sanger and Nanopore sequencing, the 8228-nucleotide-long genomic sequence of AiV (OQ121963) was determined and confirmed to belong to genotype A. The full-length genome of OQ121963 consisted of a 7296 nt open reading frame (ORF) that encodes a single polyprotein, and 5' UTR (676 nt) and 3' UTR (256 nt) at 5' and 3' ends, respectively. The ORF consisted of leader protein (L), structural protein P1 (VP0, VP1, and VP3), and nonstructural protein P2 (2A, 2B, and 2C) and P3 (3A, 3B, 3C, and 3D). The secondary structure analysis of the 5' UTR identified only stem-loop C (SL-C) and not SL-A and SL-B. The variable region of the AiV genome was analyzed by MegAlign Pro and reconfirmed by SimPlot analysis using 16 AiV whole genomes known to date. Among the entire regions, structural protein region P1 showed the lowest amino acid identity (96.07%) with reference sequence AB040749 (originated in Japan; genotype A), while the highest amino acid identity (98.26%) was confirmed in the 3D region among nonstructural protein region P2 and P3. Moreover, phylogenetic analysis of the WGS of OQ121963 showed the highest homology (96.96%) with JX564249 (originated in Taiwan; genotype A) and lowest homology (90.14%) with DQ028632 (originated in Brazil; genotype B). Therefore, the complete genome characterization of OQ121963 and phylogenetic analysis of the AiV conducted in this study provide useful information allowing to improve diagnostic tools and epidemiological studies of AiVs.

Effects of L-Type Voltage-Gated Calcium Channel (LTCC) Inhibition on Hippocampal Neuronal Death after Pilocarpine-Induced Seizure.

Epilepsy, marked by abnormal and excessive brain neuronal activity, is linked to the activation of L-type voltage-gated calcium channels (LTCCs) in neuronal membranes. LTCCs facilitate the entry of calcium (Ca2+) and other metal ions, such as zinc (Zn2+) and magnesium (Mg2+), into the cytosol. This Ca2+ influx at the presynaptic terminal triggers the release of Zn2+ and glutamate to the postsynaptic terminal. Zn2+ is then transported to the postsynaptic neuron via LTCCs. The resulting Zn2+ accumulation in neurons significantly increases the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, contributing to reactive oxygen species (ROS) generation and neuronal death. Amlodipine (AML), typically used for hypertension and coronary artery disease, works by inhibiting LTCCs. We explored whether AML could mitigate Zn2+ translocation and accumulation in neurons, potentially offering protection against seizure-induced hippocampal neuronal death. We tested this by establishing a rat epilepsy model with pilocarpine and administering AML (10 mg/kg, orally, daily for 7 days) post-epilepsy onset. We assessed cognitive function through behavioral tests and conducted histological analyses for Zn2+ accumulation, oxidative stress, and neuronal death. Our findings show that AML's LTCC inhibition decreased excessive Zn2+ accumulation, reactive oxygen species (ROS) production, and hippocampal neuronal death following seizures. These results suggest amlodipine's potential as a therapeutic agent in seizure management and mitigating seizures' detrimental effects.

Effects of Pyruvate Kinase M2 (PKM2) Gene Deletion on Astrocyte-Specific Glycolysis and Global Cerebral Ischemia-Induced Neuronal Death.

Ischemic stroke is caused by insufficient blood flow to the brain. Astrocytes have a role in bidirectionally converting pyruvate, generated via glycolysis, into lactate and then supplying it to neurons through astrocyte-neuron lactate shuttle (ANLS). Pyruvate kinase M2 (PKM2) is an enzyme that dephosphorylates phosphoenolpyruvate to pyruvate during glycolysis in astrocytes. We hypothesized that a reduction in lactate supply in astrocyte PKM2 gene deletion exacerbates neuronal death. Mice harboring a PKM2 gene deletion were established by administering tamoxifen to Aldh1l1-CreERT2; PKM2f/f mice. Upon development of global cerebral ischemia, mice were immediately injected with sodium l-lactate (250 mg/kg, i.p.). To verify our hypothesis, we compared oxidative damage, microtubule disruption, ANLS disruption, and neuronal death between the gene deletion and control subjects. We observed that PKM2 gene deletion increases the degree of neuronal damage and impairment of lactate metabolism in the hippocampal region after GCI. The lactate administration groups showed significantly reduced neuronal death and increases in neuron survival and cognitive function. We found that lactate supply via the ANLS in astrocytes plays a crucial role in maintaining energy metabolism in neurons. Lactate administration may have potential as a therapeutic tool to prevent neuronal damage following ischemic stroke.

Assessing the Removal Efficiency of Murine Norovirus 1, Hepatitis A Virus, and Human Coronavirus 229E on Dish Surfaces Through General Wash Program of Household Dishwasher.

The performance of dishwashers in removing live viruses is an important informative value in practical applications. Since foodborne viruses are present in contaminated food surfaces and water environments. Insufficient washing of dishes typically makes a carrier of foodborne viruses. Dishwashers have shown excellent performance in removing bacterial pathogens, but very limited reports related to eliminate foodborne viruses on contaminated dish surfaces. Here, murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and human coronavirus 229E (HCoV-229E) were experimentally inoculated on the dish surfaces (plate, rice bowl, and soup bowl). Plaque assay, 50% tissue culture infectious dose (TCID50), and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to determine their removal efficiency of them through the general wash program of household dishwashers. Using titration assay, MNV-1 and HAV were reduced by 7.44 and 6.57 log10 PFU/dish, and HCoV-229E was reduced by 6.43 log10 TCID50/dish through the general wash program, achieving a ≥ 99.999% reduction, respectively. Additionally, RT-qPCR results revealed that viral RNA of MNV-1 and HCoV-229E reduced 5.02 and 4.54 log10 genome copies/dish; in contrast, HAV was not detected on any dish surfaces. This study confirmed the performance of household dishwashers in removing pathogenic live viruses through the general wash program. However, residual viral RNA was not sufficiently removed. Further studies are needed to determine whether the viral RNA can be sufficiently removed using combination programs in household dishwashers.

Viability of SARS-CoV-2 on lettuce, chicken, and salmon and its inactivation by peracetic acid, ethanol, and chlorine dioxide.

Since the first SARS-CoV-2 outbreak in Wuhan, China, there has been continued concern over the link between SARS-CoV-2 transmission and food. However, there are few studies on the viability and removal of SARS-CoV-2 contaminating food. This study aimed to evaluate the viability of SARS-CoV-2 on food matrices, depending on storage temperature, and inactivate the virus contaminating food using disinfectants. Two SARS-CoV-2 strains (L and S types) were used to contaminate lettuce, chicken, and salmon, which were then stored at 20,4 and -40 °C. The half-life of SARS-CoV-2 at 20 °C was 3-7 h but increased to 24-46 h at 4 °C and exceeded 100 h at -40 °C. SARS-CoV-2 persisted longer on chicken or salmon than on lettuce. Treatment with 70% ethanol for 1 min inactivated 3.25 log reduction of SARS-CoV-2 inoculated on lettuce but not on chicken and salmon. ClO2 inactivated up to 2 log reduction of SARS-CoV-2 on foods. Peracetic acid was able to eliminate SARS-CoV-2 from all foods. The virucidal effect of all disinfectants used in this study did not differ between the two SARS-CoV-2 strains; therefore, they could also be effective against other SARS-CoV-2 variants. This study demonstrated that the viability of SARS-CoV-2 can be extended at 4 and -40 °C and peracetic acid can inactivate SARS-CoV-2 on food matrices.

Stability and inactivation of SARS-CoV-2 on food contact surfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 269 million people and killed more than 5.3 million people worldwide. Although fomite transmission of SARS-CoV-2 has been continuously reported, few studies have been conducted on food contact surfaces. Therefore, this study aimed to investigate the viability of coronaviruses on food contact surfaces and to remove SARS-CoV-2 contaminated on food contact surfaces with disinfectants. At 20 °C, SARS-CoV-2 was inactivated within 48 h on all food contact surfaces. At 4 °C, it was inactivated at 48 h on kraft paper and 96 h on parchment paper, but it was viable up to 5 days in low-density polyethylene (LDPE). At -20 °C, SARS-CoV-2 did not decrease by even 1 log on all food contact surfaces until 5 days. Treatment with 70% ethanol or 1000 ppm sodium hypochlorite for 5 min was sufficient to completely remove SARS-CoV-2 from 6 food contact surfaces. Similarly, UV-C irradiation at 60 mJ/cm2 eliminated SARS-CoV-2 contaminated on food contact surfaces. Also, the wiping test showed that even wiping an area contaminated with SARS-CoV-2 with a cloth moistened with 70% ethanol or 1000 ppm sodium hypochlorite, it took 5 min to inactivate the virus. Our findings suggested that SARS-CoV-2 contaminated on food contact surfaces in local retail may be viable enough to be transported home. However, if the type and method of use of the disinfectant suggested in this study are followed, it is possible to sufficiently control the fomite transmission of SARS-CoV-2 through food contact surfaces at home.

Combination of paper membrane-based filtration and ultrafiltration to enhance the detection of MNV, HAV, and HCoV from soil-rich post-washing water.

Risk-assessing and controlling virus transmission from soil-rich post-washing water (PWW) are crucial during harvesting raw vegetables. However, viruses are normally difficult to concentrate because of their low concentrations and complex backgrounds. Here, ultrafiltration (UF), virus adsorption-elution (VIRADEL), and optimized paper filtration-coupled ultrafiltration (PFC-UF) methods were employed to evaluate the recovery of non-enveloped murine norovirus (MNV-1), hepatitis A virus (HAV), and enveloped human coronavirus 229E (HCoV-229E) from soil-rich PWW. Among the three methods, PFC-UF outperformed the other methods in the recovery of viruses from PWW with soil content. Under the highest soil condition with virus seeded at a titer of 102 plaque-forming unit (PFU) or TCID50, the PFC-UF method exhibited an exceedingly consistent recovery rate of 78.8 ± 13.3 (MNV-1) and 44.4 ± 25.2% (HAV). However, the recovery of enveloped HCoV-229E was inferior to non-enveloped viruses. Overall, PFC-UF provided a reliable method for recovering viruses in soil-rich PWW.

Prevalence and phylogenetic analysis of human enteric emerging viruses in porcine stool samples in the Republic of Korea.

Emerging infectious diseases (EID) in humans and animals are proving to be a serious health concern. This study investigated the prevalence of emerging or re-emerging human enteric viruses in porcine stools and swabs. Eleven enteric EID viruses were selected as target viruses for the current study and ranked based on their impact on public health and food safety: enterovirus (EV), hepatitis E virus, norovirus GI and GII, sapovirus (SaV), adenovirus (AdV), astrovirus, rotavirus, hepatitis A virus, aichivirus, and bocavirus. Using real-time RT-PCR or real-time PCR, EID viruses were detected in 129 (86.0%) of 150 samples. The most prevalent virus was EV, which was detected in 68.0% of samples, followed by AdV with a detection rate of 38.0%. In following sequencing and phylogenetic analyses, 33.0% (58/176) of the detected viruses were associated with human enteric EID viruses, including AdV-41, coxsackievirus-A2, echovirus-24, and SaV. Our results show that porcine stools frequently contain human enteric viruses, and that few porcine enteric viruses are genetically related to human enteric viruses. These findings suggest that enteric re-emerging or EID viruses could be zoonoses, and that continuous monitoring and further studies are needed to ensure an integrated "One Health" approach that aims to balance and optimize the health of humans, animals, and ecosystems.

Improvement of nested reverse transcription-polymerase chain reaction (RT-PCR) with high specificity and sensitivity detection of sapovirus in food matrix.

Sapovirus (SaV) is a causative agent of human gastroenteritis in both community outbreaks and sporadic cases worldwide. Shellfish accumulate a variety of pathogens during filter feeding. In particular, the contamination of shellfish by SaV has caused several outbreaks. As reported previously, nested RT-PCR (nRT-PCR) has been widely used in clinical samples, but has not proven suitable for food samples, such as oysters. This study aimed to identify a primer set for the detection of SaV with high specificity and sensitivity in food samples. To accomplish this, primers were improved in RNA-dependent RNA polymerase (RdRp) regions of SaV whole genome sequences. The sensitivity of the improved nRT-PCR was 100-1000 times higher than that of previous nRT-PCR and > 10 times higher than that of the previous real-time RT-PCR assay. Notably, cross-reaction with other viruses or food matrices was not observed by the specificity test. This study improved the reliable primer set to detect SaV in various food matrices with high sensitivity.