miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets. It has caused huge economic losses to animal husbandry worldwide. Current commercial PEDV vaccines do not provide enough protection against variant and evolved virus strains. No specific drugs are available to treat PEDV infection. The development of more effective therapeutic anti-PEDV agents is urgently needed. Our previous study suggested that porcine milk small extracellular vesicles (sEV) facilitate intestinal tract development and prevent lipopolysaccharide-induced intestinal injury. However, the effects of milk sEV during viral infection remain unclear. Our study found that porcine milk sEV, which was isolated and purified by differential ultracentrifugation, could inhibit PEDV replication in IPEC-J2 and Vero cells. Simultaneously, we constructed a PEDV infection model for piglet intestinal organoids and found that milk sEV also inhibited PEDV infection. Subsequently, in vivo experiments showed that milk sEV pre-feeding exerted robust protection of piglets from PEDV-induced diarrhea and mortality. Strikingly, we found that the miRNAs extracted from milk sEV inhibited PEDV infection. miRNA-seq, bioinformatics analysis, and experimental verification demonstrated that miR-let-7e and miR-27b, which were identified in milk sEV targeted PEDV N and host HMGB1, suppressed viral replication. Taken together, we revealed the biological function of milk sEV in resisting PEDV infection and proved its cargo miRNAs, miR-let-7e and miR-27b, possess antiviral functions. This study is the first description of the novel function of porcine milk sEV in regulating PEDV infection. It provides a better understanding of milk sEV resistance to coronavirus infection, warranting further studies to develop sEV as an attractive antiviral.

Rural-Urban Differences in Physical Fitness and Overweight Prevalence of Children and Adolescents from Central South China.

PURPOSE: This present study aims to determine the rural-urban differences in physical fitness and overweight prevalence among children and adolescents from Central South China. METHODS: All the original parameters of physical fitness indicators for 216,718 participants aged from 7 to 15 years old were obtained from the website of the Chinese National Student Physical Fitness Database and were analyzed by SPSS. RESULTS: (1) Children and adolescents from rural areas were observed to have a more adverse physical fitness ratio, and the urban-rural differences were identified in each physical fitness indicator. (2) Rural areas had a higher overweight prevalence (p < 0.01). When compared to urban cities, overweight students from rural towns measured a significantly poorer cardiorespiratory and muscular fitness in primary school (p < 0.05), while the result in middle school was showed the opposite. (3) Rural-urban residence and sex were the moderately correlated factors for muscular fitness among overweight students. CONCLUSIONS: Urban children and adolescents in central south China had an overall healthier profile than their rural peers, particularly in overweight groups. The government and related functional departments should take the factors of rural-urban residence and sex of students into consideration when building a state strategy and interventions to promote physical activity and health.

SARS-CoV-2-Specific Adaptive Immunity in COVID-19 Survivors With Asthma.

Background: Asthma patients potentially have impaired adaptive immunity to virus infection. The levels of SARS-CoV-2-specific adaptive immunity between COVID-19 survivors with and without asthma are presently unclear. Methods: COVID-19 survivors (patients with asthma n=11, with allergies n=8, and COVID-19 only n=17) and non-COVID-19 individuals (asthmatic patients n=10 and healthy controls n=9) were included. The COVID-19 patients were followed up at about 8 months and 16 months after discharge. The clinical characteristics, lymphocyte subsets, memory T cells, and humoral immunity including SARS-CoV-2 specific antibodies, SARS-CoV-2 pseudotyped virus neutralization assay, and memory B cells were analyzed in these subjects. Results: The strength of virus-specific T cell response in COVID-19 survivors was positively correlated with the percentage of blood eosinophils and Treg cells (r=0.4007, p=0.0188; and r=0.4435, p=0.0086 respectively) at 8-month follow-up. There were no statistical differences in the levels of SARS-CoV-2-specific T cell response between the COVID-19 survivors with, and without, asthma. Compared to those without asthma, the COVID-19 with asthma survivors had higher levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) at the 8-month follow-up (p<0.05). Moreover, the level of NAbs in COVID-19 survivors was positively correlated with the percentage of Treg and cTfh2 cells (r=0.5037, p=0.002; and r=0.4846, p=0.0141), and negatively correlated with the percentage of Th1 and Th17 cells (r=-0.5701, p=0.0003; and r=-0.3656, p=0.0308), the ratio of Th1/Th2, Th17/Treg, and cTfh1/cTfh2 cell (r=-0.5356, r=-0.5947, r=-0.4485; all p<0.05). The decay rate of NAbs in the COVID-19 survivors with asthma was not significantly different from that of those without asthma at 16-month follow-up. Conclusion: The level of SARS-CoV-2-specific NAbs in COVID-19 survivors with asthma was higher than that of those without asthma at 8-month follow-up. The SARS-CoV-2-specific T cell immunity was associated with blood eosinophils and Treg percentages. The SARS-CoV-2-specific humoral immunity was closely associated with cTfh2/cTfh1 imbalance and Treg/Th17 ratio. According to the findings, asthmatic patients in COVID-19 convalescent period may benefit from an enhanced specific humoral immunity, which associates with skewed Th2/Th1 and Treg/Th17 immune.

SARS-CoV-2-Specific Adaptive Immunity in COVID-19 Survivors With Asthma

Background Asthma patients potentially have impaired adaptive immunity to virus infection. The levels of SARS-CoV-2-specific adaptive immunity between COVID-19 survivors with and without asthma are presently unclear. Methods COVID-19 survivors (patients with asthma n=11, with allergies n=8, and COVID-19 only n=17) and non-COVID-19 individuals (asthmatic patients n=10 and healthy controls n=9) were included. The COVID-19 patients were followed up at about 8 months and 16 months after discharge. The clinical characteristics, lymphocyte subsets, memory T cells, and humoral immunity including SARS-CoV-2 specific antibodies, SARS-CoV-2 pseudotyped virus neutralization assay, and memory B cells were analyzed in these subjects. Results The strength of virus-specific T cell response in COVID-19 survivors was positively correlated with the percentage of blood eosinophils and Treg cells (r=0.4007, p=0.0188;and r=0.4435, p=0.0086 respectively) at 8-month follow-up. There were no statistical differences in the levels of SARS-CoV-2-specific T cell response between the COVID-19 survivors with, and without, asthma. Compared to those without asthma, the COVID-19 with asthma survivors had higher levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) at the 8-month follow-up (p<0.05). Moreover, the level of NAbs in COVID-19 survivors was positively correlated with the percentage of Treg and cTfh2 cells (r=0.5037, p=0.002;and r=0.4846, p=0.0141), and negatively correlated with the percentage of Th1 and Th17 cells (r=-0.5701, p=0.0003;and r=-0.3656, p=0.0308), the ratio of Th1/Th2, Th17/Treg, and cTfh1/cTfh2 cell (r=-0.5356, r=-0.5947, r=-0.4485;all p<0.05). The decay rate of NAbs in the COVID-19 survivors with asthma was not significantly different from that of those without asthma at 16-month follow-up. Conclusion The level of SARS-CoV-2-specific NAbs in COVID-19 survivors with asthma was higher than that of those without asthma at 8-month follow-up. The SARS-CoV-2-specific T cell immunity was associated with blood eosinophils and Treg percentages. The SARS-CoV-2-specific humoral immunity was closely associated with cTfh2/cTfh1 imbalance and Treg/Th17 ratio. According to the findings, asthmatic patients in COVID-19 convalescent period may benefit from an enhanced specific humoral immunity, which associates with skewed Th2/Th1 and Treg/Th17 immune.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

The precision of epidemiological investigation of COVID-19 transmission in Shanghai, China.

BACKGROUND: Shanghai had a local outbreak of COVID-19 from January 21 to 24. Timely and precise strategies were taken to prevent further spread of the disease. We discussed and shared the experience of COVID-19 containment in Shanghai. PROCESS: The first two patients worked at two hospitals but no staff from the two hospitals were infected. The suspected case and his two close contacts were confirmed to be infected within 12 h. The testing rate of individuals was low. The scope of screening was minimized to two related districts and the close contact tracing was completed within 12 h, which were precise and cost-effective. CONCLUSIONS: Active monitoring, precise epidemiological investigation and timely nucleic acid testing help discover new cases, minimize the scope of screening, and interrupt the transmission.