Development and evaluation of a time-saving RT-qRPA method for the detection of genotype 4 HEV presence in raw pork liver.

Hepatitis E virus (HEV) is a zoonotic pathogen spreading worldwide. Pig was known as its first and main animal reservoir. In China, pork consumption is very large and the risk of potential HEV contamination should not be underestimated. The present study aims to develop a quantitative real-time reverse transcription combining recombinase polymerase amplification assay (RT-qRPA) for the rapid detection of HEV RNA presence in raw pork liver on the Jinzhou markets in China. Methods: the specific primers and probes for RT-qRPA assay were designed targeting the ORF2/3 conserved region in genotype 4 swine HEV isolate (accession no. DQ279091.2) according to the TwistDx manual instructions. The specificity, sensitivity and reproducibility evaluations of the RT-qRPA method were subsequently conducted in assessing agreement with the standard RT-qPCR method. Results: the qRPA method step exhibited the obvious time-saving advantage which worked under the isothermal condition at 39 °C within about 30 min to complete the run while the compared standard qPCR method in the same cycle took almost 60 min to do. Both methods could exclusively detect the HEV genome equivalents from the quantified HEV-VLPs spiked samples. And both methods shared the same limit of detection (LOD) that was estimated at 1.25 × 103 genome equivalents copies/g spiked sample by the probit analysis. The recovery rate of HEV-VLPs reached a range of 9.56-14.65% by the RT-qRPA method which was higher than that of 1.34-2.34% by the standard RT-qPCR method. The detected HEV RNA positive rate in the field was 1.8% (1 out of 55) by both methods under Cohen's kappa statistic accessing with perfect agreement (κ = 1.00, p < 0.0005). The viral load in positive sample detected by the RT-qRPA method was estimated at 2.2125 × 105 genome copies/g pork liver sample. Conclusions, the present reported RT-qRPA method mainly targeting genotype 4 HEV is a rapid and reliable method. Its time-saving quality offers a promising for the development of a portable tool used in the routine monitoring of HEV contamination in the field.

Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA.

Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at -20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay.

A recombinant polypeptide from velvet antler of Cervus nippon Temminck exhibits similar immunomodulatory effects as its natural counterpart.

CONTEXT: Velvet antler (VA) is recognized as one of the most important Chinese traditional medical herbs. To date, the immunoactivity of the single component of VA is rarely studied though its compound extracts have been well analyzed. OBJECTIVE: The current study was designed to evaluate the immunomodulatory effects of a recombinant polypeptide (rVAP32) based on the VA of the sika deer by comparison with its natural counterpart (nVAP32). MATERIALS AND METHODS: Splenocytes proliferation and NK-cell cytotoxicity assay was evaluated by the WST-8 colorimetric method. CD4+/CD8+ cell subpopulations regulation was screened by the flowcytometry method and the Th1 or Th2-related cytokine production was measured by ELISA. RESULTS: In vitro tests showed that both rVAP32 and nVAP32 could significantly stimulate splenocytes proliferation and enhance the NK-cell cytotoxicity and CD4+/CD8+ cell subpopulations when compared with the irrelevant peptide and blank control groups. Also, they demonstrated a significant capacity in up- and down-regulating the expression of Th1- and Th2-related cytokines, respectively. There is no statistically significant difference found between the rVAP32 tested group and nVAP32 control group. DISCUSSION AND CONCLUSION: The results obtained herein indicate that rVAP32 has the similar immunomodulatory effects on the immune system of mice as its counterpart nVAP32 in vitro. The further test in vivo is qualified and rVAP32 is promised for developing a new biopharmaceutical product as a substitute for nVAP32.

Development and evaluation of a RT-LAMP assay for rapid detection of hepatitis E virus from shellfish.

Hepatitis E virus (HEV) has becoming a well known zoonotic enteric pathogen and circulated widely inter human-animal-water-food. Generally, detection of the virus has relied on conventional reverse transcription-PCR (RT-PCR) and TaqMan/SYBR quantitative real-time RT-PCR (RT-qPCR), but these tools are usually disadvantages in time-consuming and expensive instruments required. In the present study, we report here on the development of a one-step single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of HEV contamination in shellfish. The amplification is completed under the isothermal condition (63 °C) for 60 min, and can be visually evaluated by staining at a time in about 1h. In addition, a total of 315 shellfish (80 Anadara granosa, 115 Scapharca subcrenata and 120 Ruditapes philippinarum) collected monthly from the Jinzhou coastal estuary of China Bohai gulf were investigated for HEV contamination by the RT-LAMP compared with a standard RT-qPCR. It was found that genotype 4 HEV was detected in all three species of shellfish sampled using the RT-LAMP assay and was in accordance with RT-qPCR detection of HEV in shellfish. Summarily, our results indicate that the RT-LAMP is a rapid, specific, sensitive and reliable method. This method offers a new tool for the routine monitoring of HEV contamination in shellfish or its harvesting waters in field.

Surveillance of hepatitis E virus contamination in shellfish in China.

BACKGROUND: Hepatitis E virus (HEV) has been confirmed to be a zoonotic virus of worldwide distribution. HEV contamination in the water environment has not been well examined in China. The objective of this study was to evaluate HEV contamination in shellfish in a coastal area of China. Such contamination would be significant for evaluating public health risks. METHODS: samples of three species shellfish were collected from thirteen points of estuarine tidal flats around the Bohai Gulf and screened for HEV RNA using an in-house nested RT-PCR assay. The detected HEV-positive samples were further verified by gene cloning and sequencing analysis. RESULTS: the overall HEV-positive detection rate is approximately 17.5% per kilogram of shellfish.  HEV was more common among S. subcrenata (28.2%), followed by A. granosa (14.3%) and R. philippinarum (11.5%). The phylogenetic analysis of the 13 HEV strains detected revealed that gene fragments fell into two known 4 sub-genotypes (4b/4d) groups and another unknown group. CONCLUSIONS: 13 different sub-genotype 4 HEVs were found in contaminated shellfish in the Bohai Gulf rim. The findings suggest that a health risk may exist for users of waters in the Bonhai area and to consumers of shellfish.  Further research is needed to assess the sources and infectivity of HEV in these settings, and to evaluate additional shellfish harvesting areas.

Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice.

Hepatitis E virus (HEV) as a recognized zoonotic pathogen has posed global burden on public health, which is exacerbated by lack of efficient vaccine. In this study, we constructed a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 that expresses and displays the hepatitis E virus antigen ORF2 utilizing an ice uncleation protein-based anchor system. After oral vaccination of BALB/c mice, significantly higher levels of ORF2-specific mucosal IgA and serum IgG were detected and cellular immunity was also induced. These findings further support that L. lactis-based HEV antigen vaccines could be used for human and animal protection against infection.

Immunomodulatory effects of a 3.2kDa polypeptide from velvet antler of Cervus nippon Temminck.

The objective of this study was to evaluate the immunomodulatory effects of a native 3.2kDa polypeptide of velvet antler from sika deer (nVAP32) on BALB/c mice immunocytes. In vitro tests showed that nVAP32 significantly stimulated splenocyte proliferation and enhanced the NK cytotoxicity and CD4(+)/CD8(+) cell subpopulations. Also, nVAP32 demonstrated a significant capacity in up- and down-regulating the expression of Th1- and Th2-related cytokines respectively. These results indicated that nVAP32 might have potential immunomodulatory effects on the immune system of mice and the further investigation on in vivo effects is qualified.

Wound healing by a 3.2 kDa recombinant polypeptide from velvet antler of Cervus nippon Temminck.

Velvet antler (VA) is used in traditional Chinese medicine to treat a wide range of ailments including the enhancement of wound healing. A 3.2 kDa recombinant polypeptide of VA from sika deer was purified and compared to native polypeptides stimulation growth of NIH3T3 cells. Both stimulated growth in a dose-dependent manner (10-100 µg/ml). To study its wound healing properties, burn-wounded rats were topically administered with recombinant VA polypeptide or native polypeptide. Rats treated with 0.05 and 0.1% (w/w) polypeptides exhibited significant wound healing. As the yield of recombinant polypeptide was 40-fold higher than that of the native polypeptide, it may therefore be a useful biopharmaceutical.