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Abstract@#Introduction: Atherosclerosis is a chronic inflammatory disease initiated by the accumulation of macrophage derived foam cells in the intima layer of artery. In mice model of atherosclerosis, murine norovirus-4 has been shown to accelerate atherogenesis. In cells, lipid biometabolism is regulated by peroxisome proliferator activated receptor γ (PPARγ). Since PPARγ is predominantly expressed in macrophages and mice macrophages are MNV-1 proliferation-permissive host, we hypothesised that PPARγ ligands may regulate atherogenesis. Methods: MNV-1 was generated via RNA-based recovery system and used to infect the RAW 264.7 cells, then subjected to oxidized low-density lipoprotein (oxLDL)-loaded and treated with ciglitazone or 15-deoxy-Delta(12,14)-PGJ(2)(15d-PGJ2). Foam cell formation was evaluated and the MNV-1 infection in all treatments was confirmed using virus titration (50% tissue culture infective dose; TCID50) and polymerase chain reaction (PCR). Results: Increment of lipid droplets was observed in all oxLDL treatment involving MNV-1 infection, ciglitazone or 15d-PGJ2 in the cytosol of RAW 264.7 cells over time compared to non-oxLDL treated cells. From the cholesterol ester (CE) content analysis amongst the oxLDL-loaded cells however, we found MNV-1 did not elicit increment of CE content. Treatment with 15d-PGJ2 resulted in increase of the CE content in oxLDL-treated cells. Interestingly, MNV-1 and ciglitazone had synergistic effect in reducing the CE content in oxLDL-treated cells. Conclusion: oxLDL stimulates foam cells formation in RAW 264.7 cells. However, MNV-1 infection did not contribute to RAW 264.7 cells derived-foam cells formation. On the other hand, 15d-PGJ2 promotes foam cells formation whilst ciglitazone inhibits the formation of foam cells derived from MNV-1-infected macrophages.
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Abstract This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.
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Objective To analyze the effect of transport and storage conditions on the detection of pathogenic nucleic acid MHV, Reo-3, MNV in laboratory mouse cecal contents samples. Methods MHV, Reo-3 and MNV were mixed with mouse cecal contents and used as reference samples,respectively. They were placed in the lysis buffer of RNA extraction reagent(buffer AVL)or normal saline, and stored at 4℃ and room temperature(22℃-25℃). RNA of these samples was extracted at 1,2,3,7,and 14 days. Then the amount of nucleic acid in samples was detected by real-time fluorescence quantitative PCR. Results A greater decrease of the amount of nucleic acid was observed when the samples were placed in normal saline than that kept in buffer AVL. The amount of nucleic acid in samples stored at 4℃ was found to be higher than that stored at 25℃ room temperature. The amount of nucleic acid in the samples which were kept in buffer AVL at 4℃ for 3 days was higher than 50%,still detectable in the samples kept for 7 days,and undetectable at 14 days. Conclusions Mouse cecal content samples are preferably stored in the lysis buffer of RNA extraction reagent and transported at 4℃ for the detection of MHV, Reo-3, and MNV nucleic acid. It is better to complete the detection test within 3 days.
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Abstract This study aimed to evaluate the elution-concentration methodology based on skimmed milk flocculation from three varieties of tomatoes (Solanum lycopersicum L. [globe], Solanum lycopersicum var. cerasiforme [cherry] and hybrid cocktail [grape tomato]) for further monitoring of field samples. Spiking experiments were performed to determine the success rate and efficiency recovery of human norovirus (NoV) genogroup II, norovirus murine-1 (MNV-1) used as sample process control virus and human adenovirus (HAdV). Mean values of 18.8%, 2.8% and 44.0% were observed for NoV GII, MNV-1 and HAdV, respectively with differences according to the types of tomatoes, with lower efficiency for cherry tomatoes. Analysis of 90 samples, obtained at commercial establishments in the metropolitan region of Rio de Janeiro State, revealed 4.5% positivity for HAdV. Bacterial analysis was also performed with no detection of Salmonella spp., L. monocytogenes and fecal coliforms. Data demonstrated that the skimmed milk flocculation method is suitable for recovering HAdV from tomatoes and highlights the need for considering investigation in order to improve food safety.
Subject(s)
Animals , Cattle , Viruses/isolation & purification , Solanum lycopersicum/chemistry , Milk/chemistry , Food Microbiology/methods , Fruit/virology , Viruses/classification , Viruses/genetics , Solanum lycopersicum/classification , Solanum lycopersicum/virology , Flocculation , Food Microbiology/instrumentation , Fruit/classification , Fruit/chemistryABSTRACT
Human norovirus (NoV) is a member of the calicivirus family, can cause nausea, vomiting, abdominal pain and diarrhea as the main clinical symptoms of acute gastroenteritis. Human norovirus infection can be popular in the world, all ages, causing serious burden of disease. Due to the lack of suitable small animal models, there is still a lack of understanding of human immune to norovirus infection and pathogenesis. Murine norovirus (MNV) was originally isolated in immune deficient mice, and causes infection and epidemic in mice. MNV provides an alternative model to study human norovirus infection and the host intestinal immune mechanism. This article will elaborate on two aspects of innate immunity and adaptive immunity.
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Objective To establish a real-time fluorescent quantitative PCR ( FQ-PCR ) method for detection of murine norovirus ( MNV) in laboratory mouse and provide the basis for establishment of a standard detection method for MNV.Methods Specific primers were designed and MNV DNA standards were prepared according to the MNV genome sequences published on NCBI .The specificity, sensitivity, repeatability and stability of the established Q-PCR method were tested.The established Q-PCR method was applied to detect 766 mouse caecum content samples to explore preliminarily the infection status of laboratory mice in Beijing .Results No cross reaction showed in human norovirus and feline calicivirus with the established Q-PCR method.The sensitivity was up to 10 copies/μL.The coefficient of variation ( CV) of intra-assay and inter-assay was less than 2%.There were 301 positive cases detected in the 766 samples of laboratory mice.Conclusions The established FQ-PCR method is accurate and effective with high specificity , sensitivity and repeatabiliy in the quantitative detetion of nucleic acid , and can be applied to rapidly and quantitatively screen MNV in laboratory mice .
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Objective To investigate the natural infection status of murine norovirus ( MNV) in laboratory mice in Shanghai area and isolate MNV from mouse cecal feces .Methods To collect cecal contents and serum samples from 319 specific pathogen-free ( SPF) mice coming from different research institutions .Reverse transcription-polymerase chain reaction ( RT-PCR) and enzyme linked immunosorbent assay ( ELISA) were used to detect MNV infection in the mice , re-spectively.The positive stool samples were diluted and filtered through 0.22 μm membrane, inoculated into RAW 264.7 cells, and then identified by RT-PCR.Results There were 95 positive results in the 319 cecal samples by RT-PCR, and the positive rate was 29.78%.Among 180 serum samples which were tested by RT-PCR, 70 samples were positive by ELISA, and the positive rate was 38.89%.The infected RAW 264.7 cells showed cytopathic effect ( CPE) within 72 h. After 3 times of freezing and thawing , RT-PCR obtained a 187 bp band.Conclusions The results from the present study show that there is a high natural infection rate of MNV in laboratory mice in Shanghai area , and the strict breeding manage-ment must be strengthened .
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Objective To establish an indirect immunofluorescence assay for detection of murine norovirus ( MNV) .Methods Mouse leukaemic monocyte macrophage cell line RAW 264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells , and to make antigen glass slides .BALB/c mice were gavaged with MNV-1 (107 TCID50) and infected sera were collected as positive control .The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay ( IFA ) .80 serum samples were tested using the two methods , IFA and ELISA, and the discrepant samples were validated by Western blotting .Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred.IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection , reaching a maximum antibody concentration at 4 weeks after infection , and maintained a stable level later .The mouse serum at four weeks after MNV-1infection was used as positive quality control . Among the 80 serum samples , 27 positive and 53 negative cases were detected by IFA method , and 32 positive and 48 negative cases were detected by ELISA .The five discrepant samples were verified by Western blotting , resulted in 3 positive and 2 negative cases . The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally .IFA and ELISA detection can be selected as initial screening measures , and use Western blot assay to verify the discrepant samples .
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This study investigated the recovery and absorption rates of murine norovirus, a surrogate for human norovirus, by using NanoCeram(R) filters which served as a tool for recovering viruses. In the study, two types of NanoCeram(R) filters were employed: one was a cartridge type and the other was a disc type (phi 47 mm) whose surface area is 75 times smaller than the cartridge type. The analytical method was the real-time reverse transcription-polymerase chain reaction (RT-PCR). The study found that the average recovery rates of the cartridge type and the disc type were 30.9% and 29.5% respectively. Since these two rates were very close to each other, the adsorption rate of the cartridge type could be predicted with the disc type. Analyzing recovery and absorption rates of the disc type based on different filtered volumes showed that when the volume increased from 0.5 L to 20 L, the average recovery rate rose from 14.78% to 30.41 %, while the average absorption rate dropped from 56.33% to 10.48%. The increase in turbidity from less than 1 NTU to less than 3 NTU raised the average recovery rate from 47.23% to 82.84%.
Subject(s)
Humans , Absorption , Adsorption , NorovirusABSTRACT
Murine norovirus (MNV) is a non-enveloped virus with a positive-sense RNA genome and causes lethal infection in mice. MNV has been used as a model virus for human norovirus (NV) whose in vitro cell culture system has not been available to date since MNV and NV are genetically related. In this study, the genome replication of MNV was investigated using strand-specific RT-PCR in RAW264.7 cells. Reverse transcription (RT) using a sense primer followed by PCR showed that negative-sense RNAs were first detected in RAW264.7 cells between 6 and 9 [3 and 6] hours post infection (h.p.i.). However, these negative-sense RNAs were not detected when cells were treated with a translation inhibitor cycloheximide. Then, RT with an antisense primer followed by PCR was performed to detect positive-sense RNAs. RT-PCR results revealed that the amount of positive-sense RNAs began to increase from 9 [6] h.p.i., indicating the accumulation of the newly synthesized (+)RNA genome. Furthermore, cycloheximide abrogated the increase of newly made RNAs during MNV infection. In conclusion, strand-specific RT-PCR using a sense or antisense primer, in combination with cycloheximide treatment, enabled us to detect positive-sense and negative-sense RNAs selectively and provided a useful tool to understand the replication cycle of MNV.