Sequential infection of human norovirus and Salmonella enterica resulted in higher mortality and ACOD1/IRG1 upregulation in zebrafish larvae.

Human norovirus (HNoVs) and Salmonella are both very important foodborne pathogens with mixed infection of HNoV and Salmonella reported clinically. With the use of model organism zebrafish (Danio rerio), it was observed that the sequential infection of HNoVs and Salmonella caused lower survival rates (12.5 ± 4.2%) than the single-pathogen infection by Salmonella (31.6 ± 7.3%, P < 0.05) or HNoVs (no mortality observed). Gene expression study with the use of RT-PCR and global transcriptomic analysis revealed that the mortality of zebrafish larvae was very likely due to the harmful inflammatory responses. Specifically, it was noted that the genes encoding aconitate decarboxylase 1 (ACOD1), also known as immunoresponsive gene 1 (IRG1), were significantly upregulated in the sequentially infected zebrafish larvae. The expression of acod1 could lead to mitochondrial reactive oxygen species (ROS) production. The ROS levels were indeed higher in sequentially infected zebrafish larvae than the single-pathogen infected ones (P < 0.05). An immersion treatment of glutathione or citraconate did not affect the microbial loads of HNoVs and Salmonella but significantly reduced the ROS levels and protected the zebrafish larvae by inducing higher survival rates in the sequentially infected zebrafish larvae (P < 0.05). Taken together, this study accumulated new knowledge over the function of ACOD1/IRG1 pathway in infectious diseases, and proposed possible treatment strategies accordingly.

Use of Zebrafish Embryos To Reproduce Human Norovirus and To Evaluate Human Norovirus Infectivity Decay after UV Treatment.

This study reports an essential improvement of the method for replication of human norovirus (HNoV) with the use of zebrafish (Danio rerio) embryos. With three HNoV genotypes and P-types GII.2[P16], GII.4[P16], and GII.17[P31], we demonstrated that this tool had higher efficiency and robustness than the zebrafish larvae as reported previously. When zebrafish larvae were injected with virus (1.6 ± 0.3 log genome copies/10 larvae), a significant increase of virus genome copies was detected at 2 days postinfection (dpi; 4.4 ± 0.8 log genome copies/10 larvae, P < 0.05) and the viral loads started to decrease gradually from 3 dpi. In comparison, when the viruses were injected into the zebrafish embryos, significant virus replication was noticed from 1 dpi and lasted to 6 dpi (P < 0.05). The virus levels detected at 3 dpi had the highest mean value and the smallest variation (7.7 ± 0.2 log genome copies/10 larvae). The high levels of virus replication enabled continuous passaging for all three strains up to four passages. The zebrafish embryo-generated HNoVs showed clear patterns of binding to human histo-blood group antigens (HBGAs) in human saliva by a simple saliva-binding reverse transcription-quantitative PCR (RT-qPCR). Last, in a disinfection study, it was shown that a dose of 6 mJ/cm2 UV254 was able induce a >2-log reduction in HNoV infectivity for all three HNoV strains tested, suggesting that HNoVs were more UV susceptible than multiple enteric viruses and commonly used HNoV surrogates as tested before. IMPORTANCE HNoVs are a leading cause of gastroenteritis outbreaks worldwide. The zebrafish embryo tool as developed in this study serves as an efficient way to generate viruses with high titers and clean background and a straightforward platform to evaluate HNoV inactivation efficacies. It is expected that this tool will not only benefit epidemiological research on HNoV but also be used to generate HNoV inactivation parameters which are highly needed by the water treatment and food industries.

The globally re-emerging norovirus GII.2 manifests higher heat resistance than norovirus GII.4 and Tulane virus.

AIMS: To compare the heat stability of two globally prevalent human norovirus (HuNoV) strains (GII.2[P16] and GII.4[P16]) and a commonly used HuNoV surrogate, Tulane virus (TV). METHODS AND RESULTS: With the use of a newly developed zebrafish larvae platform, we measured the change of infectivity of HuNoV GII.2[P16] and GII.4[P16] toward mild heat treatment at 55°C for 5 min. TV was tested with the same experimental design. As a result, the virus infectivity measurement clearly indicated the higher heat resistance of HuNoV GII.2[P16] (no reduction) than GII.4[P16] (>0.8-log TCID50  ml-1 reduction) and TV (2.5-log TCID50  ml-1 reduction). Further exploration revealed higher virus structural stability of HuNoV GII.2 than GII.4 strains by the use of different clinical samples with different evaluation methods. CONCLUSION: The inactivation data generated from the surrogate virus TV cannot be used directly to describe the inactivation of HuNoV. The phylogenetic classification of HuNoVs may correlate with the virus stability and/or circulation dynamics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is expected to serve as an important reference when revisiting the numerous previous data evaluating HuNoV inactivation conditions in foods with the use of TV as the cultivable surrogate or with genuine HuNoV but using molecular methods. The higher resistance of NoV GII.2 strains than GII.4 strains toward inactivation treatment supplies a possible explanation for the global re-emerging of NoV GII.2 epidemic in recent years.

No Clinical Symptom Experienced after Consumption of Berry Fruits with Positive RT-qPCR Signals of Human Norovirus.

Human noroviruses (hNoVs) are the most important foodborne viruses, and soft berries are one of the most common food sources of hNoV outbreaks and contamination. This paper presents a human volunteer study in order to investigate the correlation between molecular detection results of hNoV in berries with the public health risks. The participants with diverse histo-blood group antigens (HBGAs) phenotypes were required to consume self-purchased berries and meanwhile submit aliquots of the products for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection. As a result, none of the 20 participants reported any hNoV infection-like symptoms after six independent consumptions (120 consumptions in total). In contrast, within the 68 berry samples with >1% virus recoveries, 28 samples were detected to be positive for hNoV GI and/or GII (the positive rate at 41%). All of the positive signals were below the limit of quantification (<120 genome copies/g) except one fresh strawberry sample at 252 genome copies/g. It is expected that this study would contribute to the definition of quantitative standards for risk assessment purposes in the future.

Fucoidan But Not 2'-Fucosyllactose Inhibits Human Norovirus Replication in Zebrafish Larvae.

Human noroviruses (hNoVs) cause heavy disease burden worldwide and there is no clinically approved vaccination or antiviral hitherto. In this study, with the use of a zebrafish larva in vivo platform, we investigated the anti-hNoV potentials of fucoidan (from brown algae Fucus vesiculosus) and 2'-Fucosyllactose (2'-FL). As a result, although both fucoidan and 2'-FL were able to block hNoV GII.4 virus-like particle (VLPs) from binding to type A saliva as expected, only fucoidan, but not 2'-FL, was able to inhibit the replication of hNoV GII.P16-GII.4 in zebrafish larvae, indicating the possible needs of higher molecular weights for fucosylated carbohydrates to exert anti-hNoV effect.

Influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated norovirus binding.

Bivalve molluscan shellfish such as oysters are filter feeders and are able to accumulate human noroviruses (NoVs) largely due to the presence of human histo-blood group antigens (HBGAs)-like carbohydrates in their intestine. Since the fucose contents play a key role in the binding of NoVs to HBGAs, this study intended to investigate the influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated NoV binding. On the contrary to the expected, after a treatment of the oyster digestive tissue extracts with Bifidobacterium bifidum strain JCM 1254, the binding of human NoV GII.4 virus like particles (VLPs) to the oyster digestive tissue extracts enhanced significantly (OD450 from 1.15 ± 0.05 to 1.51 ± 0.02, P < 0.001) in an in vitro direct binding assay. The accumulation of human NoV GII·P16-GII.4 also enhanced significantly in the intestine of B. bifidum JCM 1254 treated oysters from 4.27 ± 0.25 log genomic copies/g oyster digestive tissue to 5.25 ± 0.29 log genomic copies/g oyster digestive tissue (P < 0.005) as observed in an in vivo test. Correspondingly, the type A antigenicity of the oyster digestive tissue extracts enhanced (OD450 from 0.77 ± 0.04 to 1.06 ± 0.05, P < 0.01) after the treatment with B. bifidum JCM 1254. These results could be explained by the substrate specificity of the B. bifidum JCM 1254 associated fucosidases. This study identified an indirect interaction possibly happening between the bacterial microbiota with human NoVs during their transmission in the food systems. We also supplied a potential strategy to mitigate the NoV contamination from shellfish, suppose bacterial strains with specified fucosidase production could be obtained in the future.