Je-Ruei Liu's research while affiliated with Academia Sinica and other places

Influenza, also known as “flu”, is an infectious disease caused by influenza viruses. Three types of influenza virus, A, B, and C, are able to infect humans. In most people, influenza causes mild symptoms, but it can also induce severe complications and death. Annual influenza vaccines are currently the main intervention used to minimize mortality and morbidity. However, vaccination frequently fails to provide adequate protection, especially in the elderly. Traditional flu vaccine targets hemagglutinin to prevent virus infection, but the constant mutation of hemagglutinin means that it is a challenge to develop vaccines quickly enough to keep up with mutations. Thus, other methods of curbing influenza incidence would be welcomed, especially for vulnerable populations. Although influenza viruses primarily infect the respiratory tract, influenza virus infection also induces intestinal dysbiosis. Through gut microbiota-derived secreted products and the circulating immune cells, gut microbiota can affect pulmonary immunity. The crosstalk between the respiratory tract and gut microbiota, termed the “gut-lung axis”, is observed in the regulation of immune responses against influenza virus infection or inflammation-induced lung damage, indicating the possibility of using probiotics to prevent influenza virus infection or alleviate respiratory symptoms. In this review, we summarize the current findings on the antiviral functions of particular probiotics and/or combinations and discuss the antiviral mechanisms and immunomodulatory activities of probiotics in vitro, in mice, and in humans. Clinical studies show probiotic supplements can provide health benefits, not only to the elderly or children with compromised immune systems, but also to young- and middle-aged adults.

Newly emerging and re-emerging viral infectious diseases cause significant economic losses in swine production. Efficacious vaccines have not yet been developed for several major swine infectious diseases, including porcine epidemic diarrhea virus (PEDV). We used the PEDV-infected Vero cell model to screen lactic acid bacteria (LAB) strains with antiviral activity. Sixty LAB strains were isolated from the feces of nursing piglets. After the elimination of LAB strains with high cytotoxicity to Vero cells, the protective effects of the remaining 6 strains against PEDV infection were determined. Vero cells pretreated with the intracellular extracts or cell wall fractions of YM22 and YM33 strains for 24 h before infection with PEDV showed significantly higher cell viabilities and lower mRNA expression of PEDV nucleocapsid (PEDV-N) than the unpretreated cells, indicating that the intracellular extracts and cell wall fractions of YM22 and YM33 possessed prophylactic effects on Vero cells against PEDV infection. PEDV-infection significantly increased the mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in Vero cells. However, pretreatment of Vero cells with the cell wall fractions of YM22 and YM33 decreased the mRNA expression of TNF-α and IL-8, which could be a mechanism associated with the protective effects of YM22 and YM33 against PEDV. Based on the biochemical characteristics and phylogenetic analyses, YM22 and YM33 were identified as Ligilactobacillus agilis (basonym: Lactobacillus agilis) and Ligilactobacillus salivarius (basonym: Lactobacillus salivarius), respectively. These findings suggest that L. agilis YM22 and L. salivarius YM33 could provide some levels of protective effects against PEDV infections.

This study aimed to investigate the potential of Bacillus licheniformis-fermented products (BLFP) and their derived antimicrobial lipopeptide, surfactin, for the prevention of coccidiosis in broilers. Broilers were fed BLFP at 1.25 and 5 g/kg under Eimeria tenella challenge. At the end of experiment (35 days), the growth performance, survival rate, cecal morphology, cecal lesion scores, oocyst-count index, and anti-coccidial index were analyzed. The effects of the BLFP-derived surfactin on oocyst sporulation and sporozoite morphology in Eimeria species were also investigated in vitro. Results showed that BLFP supplementation at 1.25 and 5 g/kg improved cecal morphology and increased the survival rate of broilers under E. tenella challenge. Supplementation with 1.25 g/kg of BLFP reduced the lesion scores in the cecum of E. tenella-challenged broilers, while the oocyst-count index was reduced in broilers given 5 g/kg of BLFP. The anti-coccidial index of the 1.25 g/kg of BLFP-treated group was greater than 160, compared with the E. tenella-challenge-only group. Furthermore, surfactin inhibited Eimeria oocyst sporulation and disrupted sporozoite morphology. These results demonstrate that BLFPs and their derived antimicrobial lipopeptide, surfactin, exhibit anti-coccidial activity in vitro and in vivo. BLFP may be used as a natural feed additive for the prevention of coccidiosis in broilers, and 1.25 g/kg can be considered the optimum dosage.

Fruits have been widely considered as the “default health foods” as they contain numerous vitamins and minerals needed to sustain human health. To fully enhance their nutritive and flavor features while prolonging their shelf lives, fermentation strategies have been utilized to prepare healthy yet readily consumable fruit products. However, albeit their innumerable health-promoting effects, a lot of people are still concerned if consuming live bacteria and their metabolites are safe for humans. In this study, the safety of a fermented multi-fruit beverage using five different fruits (kiwis, guavas, papayas, pineapples, and grapes) was investigated. Safety assessment experiments performed on the multi-fruit beverage showed that it is non-mutagenic to five strains of Salmonella typhimurium,non-cytotoxic, and is unable to induce chromosome aberrations in Chinese Hamster Ovary K1 (CHO-K1) cells, as well as non-toxic in male ICR mice compared to the water-fed control group after 28 d of feeding. Therefore, these results collectively indicated that the fermented multi-fruit beverage is safe for human consumption.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Obesity is closely associated with various metabolic disorders, including leptin resistance, which is characterized by high circulating leptin levels. Probiotics can decrease circulating leptin levels by alteration of the gut microbiota. Thus, they may have anti-obesogenic effects. In this study, the effects of administration of a probiotic bacterium, Lactobacillus rhamnosus GG (LGG), on gut microbiota and modulation of leptin resistance were evaluated in mice. Male Balb/C mice aged 7 weeks were fed either a normal diet (ND), high-fat diet (HFD), HFD supplemented with low-dose LGG (108 CFU/mouse/day), or HFD supplemented with high-dose LGG (1010 CFU/mouse/day) for 10 weeks. Significantly increased body weight, epididymal fat weight, and decreased leptin responsiveness to exogenous leptin treatment and ratio of villus height to crypt depth were observed in the HFD-fed mice compared to the ND-fed mice. Moreover, a remarkable increase in the proportion of Proteobacteria and ratio of Firmicutes/Bacteroidetes in the fecal microbiota were also observed in the HFD-fed mice. Supplementation of HFD with high-dose LGG restored exogenous leptin responsiveness, increased the ratio of villus height to crypt depth, and decreased the proportion of Proteobacteria in fecal microbiota. These findings suggest that LGG supplementation might alleviate leptin resistance caused by an HFD through the improvement of the digestive health of the host.

Swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. Previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. In this study, we used the Vero cell culture model of infection to study porcine epidemic diarrhea virus (PEDV). We screened lactic acid bacteria (LAB) with anti-PEDV potential from kefir grains, which are starter cultures used to ferment milk into kefir. Twenty-nine LAB strains were isolated and identified as Enterococcus durans, Lactobacillus kefiri, Lactococcus lactis, and Leuconostoc mesenteroides, according to 16S ribosomal RNA (rRNA) and rpoA gene sequence analyses. The anti-PEDV activities of the LAB intracellular extracts were compared, and the intracellular extracts of Ln. mesenteroides showed higher anti-PEDV activities than that of the other species. Among the Ln. mesenteroides strains, a strain designated YPK30 showed a higher growth rate than that of the other strains and was further evaluated for its anti-PEDV activity. The results showed that the intracellular extracts of Ln. mesenteroides YPK30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against PEDV in the Vero cell model. The expression levels of Type 1 interferon (IFN)-dependent genes, including Myxovirus resistance 1 (MX1) and interferon-stimulated gene 15 (ISG15), were significantly increased after treatment with intracellular extracts of Ln. mesenteroides YPK30 for 24 h. Such expression suggests that the anti-PEDV activity of Ln. mesenteroides YPK30 could be attributed to its up-regulatory effect on the expression of MX1 and ISG15 genes. These results suggested that Ln. mesenteroides YPK30 has the potential to provide some levels of host protection against PEDV infections.

Fruits have been widely considered as the default “health foods” because they contain numerous vitamins and minerals needed to sustain human health. Fermentation strategies have been utilized to enhance the nutritive and flavor features of healthy and readily consumable fruit products while extending their shelf lives. A traditional fermented multi-fruit beverage was made from five fruits including kiwi, guava, papaya, pineapple, and grape fermented by Saccharomyces cerevisiae along with lactic acid bacteria and acetic acid bacteria. The immunomodulatory properties of the fermented multi-fruit beverage, in vivo nonspecific and ovalbumin (OVA)-specific immune response experiments using female BALB/c mice were performed. Administration of the fermented multi-fruit beverage reduced the calorie intake, thus resulting in a less weight gain in mice compared to the water (placebo)-fed mice. In the nonspecific immune study model, the fermented multi-fruit beverage enhanced phagocytosis and T cell proliferation but did not affect B cell proliferation and immunoglobulin G (IgG) production. Analysis of cytokine secretion profile also revealed that the fermented multi-fruit beverage enhanced proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and T helper (Th)1-related cytokine interferon (IFN)-γ production, thus creating an immunostimulatory effect. Nonetheless, in the specific immune study model, the results showed that the fermented multi-fruit beverage decreased the production of proinflammatory cytokines IL-6 and TNF-α production in OVA-immunized mice. Moreover, it also caused a decrease in the production of anti-OVA IgG1, which was accompanied by a decrease in Th2-related cytokines IL-4 and IL-5 production and an increase in Th1-related cytokine IFN-γ production, indicating that it may have the potential to shift the immune system from the allergen‐specific Th2 responses toward Th1-type responses. The results indicate that fermented multi-fruit beverage has the potential to modulate immune responses both in a nonspecific and specific manners.

Porcine circovirus type 2 (PCV2) is a pathogenic virus that causes high rates of porcine death, resulting in severe economic losses to the swine industry. In recent years, the prevalence of PCV2d genotype infection in pigs has increased, but most commercially available vaccines were developed against the PCV2a strain and do not ensure complete protection from PCV2d. Here, we first constructed an expression vector for the antigenic ORF2-encoded capsid protein of PCV2d (pLp3050-His6-tag-capsid). We then utilized Lactobacillus plantarum to express the protein at mucosal sites in orally vaccinated mice. After transducing L. plantarum with pLp3050-His6-tag-capsid, the expressed protein could be found in cell wall and cell-free supernatant fractions by Western blotting. Using flow cytometry, we found that L. plantarum cells with surface-displayed capsid protein increased with time after SppIP induction. Finally, mice that were orally immunized 18 times with capsid-expressing L. plantarum showed increased levels of capsid-specific sIgA and virus neutralizing activity at mucosal sites, suggesting mucosal immunity had been stimulated by the vaccine. Overall, our findings demonstrate the feasibility and utility of a PCV2d-based vaccine, which may be of great value in porcine agriculture.

The difficulty of long-term management has produced a high rate of failure for obesity patients. Therefore, improving the efficacy of current obesity treatment is a significant goal. We hypothesized that combining a probiotic Lactobacillus mali APS1 intervention with dieting could improve the efficacy of obesity and hepatic steatosis treatment compared to dieting alone. Mice were fed a high-fat diet for 6 weeks and then treated with: saline + normal diet and APS1 + normal diet (NDAPS1) for 3 weeks. NDAPS1 accelerated body weight loss and reduced caloric intake and fat accumulation. The fecal microbiome showed that accelerating weight loss by NDAPS1 resulted in restoring intestinal microbiota toward a pre-obese state, with alteration of specific changes in the obesity-associated bacteria. APS1 manipulated the gut microbiome's obesity-associated metabolites, followed by regulation of lipid metabolism, enhancement of energy expenditure and inhibition of appetite. The specific hepatic metabolites induced by the APS1-manipulated gut microbiome also contributed to the amelioration of hepatic steatosis. Our results highlighted a possible microbiome and metabolome that contributed to accelerating weight loss following treatment with a combination of APS1 and dieting and suggested that probiotics could serve as a potential therapy for modulating physiological function and downstream of the microbiota.