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1.
Water Sci Technol ; 83(5): 1103-1107, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33724939

RESUMO

Noroviruses are significant seafood-borne pathogens, commonly associated with the consumption of filter feeding bivalve molluscs. Here, we report the development of a reverse transcription polymerase chain reaction (RT-PCR) method using primers based on the RNA-dependent RNA polymerase gene of norovirus genogroup II (NoV GII). Samples of bivalves were processed for the concentration of virus and extraction of RNA, followed by reverse transcription PCR. A total of 50 molluscan shellfish samples were analyzed, of which 16 samples yielded positive amplifications of norovirus nucleic acid. The PCR method described here, involving a single set of primers, is useful for rapid screening of shellfish for NoV GII.


Assuntos
Bivalves , Norovirus , Animais , Genótipo , Humanos , Norovirus/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa
2.
Int J Food Microbiol ; 344: 109089, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33662900

RESUMO

Contamination of bivalve molluscs with viruses is well recognized as a food safety risk. A microbiological criterion for norovirus (NoV) and hepatitis A virus (HAV) in shellfish, however, does not exist in the European Union currently. The aim of this study was to evaluate the contamination levels of these viruses for fluctuation over a long period (2013-2017) in oyster (n = 266) and mussel samples (n = 490) using a method based on ISO/TS 15216-1: 2013. Samples were taken at different points in the food chain, either directly post-harvest, at Dutch dispatch centers or in retail stores, from September until March of each year. Altogether, 53.1% of the mussel and 31.6% of the oyster samples tested positive for NoV RNA. Simultaneous presence of NoV GI and GII RNA was observed in 31.6% of mussel and 10.2% of oyster samples. Contamination levels in NoV positive mussel samples collected post-harvest from B-areas were significantly higher than in those collected post-harvest from A-areas, or at dispatch centers or retail stores. Levels in oysters from dispatch were significantly lower than those collected in retail stores. Ready for sale mussels and oysters contained 2.04 and 1.76 mean log10 transformed NoV genome copies/gram (gc/g), respectively. GII levels were at a constant level in ready for sale mussels throughout all sampling periods in the study. This seemed to be true for oysters as well. HAV RNA was detected in only one of the tested mussel samples (n = 392) (typed HAV 1A) and in none of the tested oyster samples (n = 228). Critical evaluation of NoV and HAV levels in shellfish can be of help for risk assessment and risk management actions.


Assuntos
Infecções por Caliciviridae/epidemiologia , Vírus da Hepatite A/isolamento & purificação , Hepatite A/epidemiologia , Norovirus/isolamento & purificação , Ostreidae/virologia , Animais , Infecções por Caliciviridae/veterinária , Cadeia Alimentar , Contaminação de Alimentos/análise , Inocuidade dos Alimentos , Hepatite A/veterinária , Vírus da Hepatite A/genética , Humanos , Países Baixos/epidemiologia , Norovirus/genética , Frutos do Mar/virologia
3.
Water Res ; 196: 116990, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33725645

RESUMO

Noroviruses (NoVs) are the leading cause of acute gastroenteritis (AGE) outbreaks. Since 2014, novel genetic variants of NoV have been continuously identified and have caused a sharp increase in the number of AGE outbreaks. The specific geographical distribution and expanding genetic diversity of NoV has posed a challenge to conventional surveillance. Here, we describe the long-term dynamic correlation between NoV distribution in sewage and in the local population through the molecular surveillance of NoV in Guangdong, 2013-2018. The relative viral loads of the GI and GII genotypes in sewage were calculated through RT-PCR. A high-throughput sequencing method and operational taxonomic unit (OTU) clustering pipeline were developed to illustrate the abundances of different genotypes and genetic variants in sewage. Our results showed that the NoV viral loads and the emergence of new variants in sewage were closely associated with NoV outbreak risks in the population. Compared with the outbreaks surveillance, the dominance of the newly emerged variants, GII.P17-GII.17 and GII.P16-GII.2, could be detected one or two months ahead in sewage of a hub city. In addition, the dynamics of pre-epidemic variants, which were rarely detected in clinics, could be captured through sewage surveillance, thus improving our understanding of the origin and evolution of these novel epidemic variants. Our data highlight that sewage surveillance could provide nearly real-time and high-throughput data on NoV circulation in the community. With the advances in sequencing techniques, the sewage surveillance system could also be extended to other related infectious diseases.


Assuntos
Infecções por Caliciviridae , Norovirus , Infecções por Caliciviridae/epidemiologia , China/epidemiologia , Cidades , Surtos de Doenças , Genótipo , Humanos , Norovirus/genética , Filogenia , Esgotos
4.
Medicine (Baltimore) ; 100(12): e25123, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33761678

RESUMO

ABSTRACT: Human norovirus (NoV) is the leading cause of acute gastroenteritis and the rapid transmission of NoV renders infection control problematic. Our study aimed to investigate viral shedding in gastroenteritis in children caused by variants of emerging norovirus strains infections.We used RNA-dependent RNA polymerase (RdRp) sequencing to measure NoV genome copies in stool to understand the relationship between the clinical manifestations and viral shedding in hospitalized patients. The near full-length NoV genome sequence was amplified via reverse transcription-polymerase chain reaction (RT-PCR) and NoV recombination was analyzed using the Recombination Analysis Tool (RAT).From January 2015 to March 2018, 77 fecal specimens were collected from hospitalized pediatric patients with confirmed NoV gastroenteritis. The NoV genotypes were GII.4 (n = 22), non-GII.4 (n = 14), GII.4 Sydney (n = 21), and GII.P16-GII.2 (n = 20). Viral load increased from days 2 to 9 from the illness onset, resulting in an irregular plateau without peaks. After day 9, the viral load declined gradually and most viral shedding in feces ceased by day 15. The average viral load was highest in GII.4 Sydney followed by GII.P16-GII.2 infections and lowest in non-GII.4 infections. GII.4 unclassified infections showed the longest viral shedding time, followed by GII.4 Sydney infections, GII.P16-GII.2 recombinant infection resulted in the shortest duration. NoVs evolved to form a group of GII.P16-GII.2 variants during the 2017 to 2018 period.The viral load and shedding period and was different in variants of NoV infections in children. High mutation rate of emerging and re-emerging variants was observed to an enhanced epidemic risk rendering continuous surveillance.


Assuntos
Infecções por Caliciviridae/virologia , Gastroenterite/virologia , Variação Genética , Norovirus/genética , Eliminação de Partículas Virais/genética , Pré-Escolar , Fezes/virologia , Feminino , Genótipo , Humanos , Lactente , Pacientes Internados/estatística & dados numéricos , Masculino , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Recombinação Genética , Taiwan , Carga Viral
5.
Talanta ; 225: 121978, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33592726

RESUMO

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.


Assuntos
Infecções por Caliciviridae/diagnóstico , Colorimetria/métodos , Gastroenterite/diagnóstico , Infecções por Caliciviridae/virologia , Colorimetria/economia , Colorimetria/instrumentação , Análise Custo-Benefício , Gastroenterite/virologia , Genótipo , Humanos , Norovirus/genética , Norovirus/fisiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Papel , RNA Viral/genética , Sensibilidade e Especificidade
6.
BMC Infect Dis ; 21(1): 54, 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435906

RESUMO

BACKGROUND: An outbreak of acute gastroenteritis occurred in a kindergarten located Shenzhen City on March 4, 2018. We were invited to investigate to the risk factors associated with this outbreak. METHODS: We conducted retrospective cohort-studies on three different groups of subjects in order to figure out the difference of incidence of acute gastroenteritis among subjects of different activities on March 2: group one consisted of people who attended the Lantern festival activities; group two consisted of children and employees who ate breakfast and bread provided by the kindergarten; and groups three consisted of children and employees who did not eat breakfast or bread provided by the kindergarten. Fecal, anal swabs, dishware swabs and hand swabs specimens were collected in the study. Bacteria known to cause acute gastroenteritis were cultured. Viruses associated with acute gastroenteritis were tested using real-time PCR. Capsid gene fragment of 557 bp of norovirus was amplified and sequenced. The phylogenetic tree was constructed with MEGA 7.0 using neighbor-joining method based on capsid gene fragment of norovirus. RESULTS: A total of 143 suspected cases were identified in this outbreak. Diarrhea happened more often in adults than in children while emesis and bellyache were more frequently found in children than in adults. Higher AGE incidence was observed in group 2, children and employees who had breakfast in the kindergarten on March 2, as well as in group 3, and among employees who eating bread involved in breakfast provided on March 2. Five anal swab specimens were positive for norovirus. All noroviruses belongs to group II.3 and have an identity more than 99%. CONCLUSION: A chef, as an asymptomatic carrier with norovirus, was the infectious resource in this outbreak. He contaminated breakfast food provided on March 2. Although morning check is implemented in kindergartens of China, employees are often excluded in morning check. Our finding highlights the importance of morning check covering employees and periodical training for cooks.


Assuntos
Desjejum , Infecções por Caliciviridae/epidemiologia , Portador Sadio/virologia , Surtos de Doenças , Manipulação de Alimentos , Gastroenterite/epidemiologia , Norovirus/genética , Escolas Maternais , Adulto , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/virologia , Criança , Pré-Escolar , China/epidemiologia , Diarreia/epidemiologia , Diarreia/virologia , Fezes/virologia , Feminino , Microbiologia de Alimentos/métodos , Gastroenterite/diagnóstico , Gastroenterite/prevenção & controle , Gastroenterite/virologia , Humanos , Incidência , Masculino , Filogenia , Quarentena/métodos , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Risco , Vômito/epidemiologia , Vômito/virologia
7.
J Infect Public Health ; 14(2): 244-248, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33493921

RESUMO

BACKGROUND: Recently, monoclonal-antibody-conjugated immunomagnetic separation (IMS) procedure combined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used for quantifying non-cultivated human noroviruses (HuNoVs). METHODS: We examined the efficacy of 27 commercially available disinfectants and a prototype against GII.4 strain HuNoV through the IMS/qRT-PCR assay. RESULTS: The average log reduction in viral titer in vitro varied among the disinfectants. The prototype was the most effective with an average log reduction of 6.86 log. CONCLUSIONS: The IMS/RT-qPCR assay is an effective method to evaluate the activities of disinfectants against GII.4 HuNoV in vitro. Further work is needed to enhance the virucidal activity of the prototype disinfectant against more resistant HuNoV strains.


Assuntos
Desinfetantes/farmacologia , Separação Imunomagnética/métodos , Norovirus/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Norovirus/genética , Norovirus/isolamento & purificação , Carga Viral , Inativação de Vírus
8.
Arch Virol ; 166(3): 905-913, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33462673

RESUMO

From 2010-2016, a total of 251 stool samples were screened for norovirus using next-generation sequencing (NGS) followed by phylogenetic analysis to investigate the genotypic diversity of noroviruses in rural and low-income urban areas in northern Brazil. Norovirus infection was detected in 19.9% (50/251) of the samples. Eight different genotypes were identified: GII.4_Sydney[P31] (64%, 32/50), GII.6[P7] (14%, 7/50), GII.17[P17] (6%, 3/50), GII.1[P33] (6%, 3/50), GII.3[P16] (4%, 2/50), GII.2[P16] (2%, 1/50), GII.2[P2] (2%, 1/50), and GII.4_New Orleans[P4] (2%, 1/50). Distinct GII.6[P7] variants were recognized, indicating the presence of different co-circulating strains. Elucidating norovirus genetic diversity will improve our understanding of their potential health burden, in particular for the GII.4_Sydney[P31] variant.


Assuntos
Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Norovirus/genética , Norovirus/isolamento & purificação , Pobreza/estatística & dados numéricos , Sequência de Bases , Brasil/epidemiologia , Estudos Transversais , Fezes/virologia , Gastroenterite/virologia , Variação Genética/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Epidemiologia Molecular , Norovirus/classificação , Filogenia , RNA Viral/genética
9.
Int J Food Microbiol ; 339: 109033, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33401188

RESUMO

An increasing number of hepatitis E virus (HEV) infections in industrialized countries have been foodborne and linked to the consumption of undercooked pork products. To date, data on the prevalence of HEV in pork products sold in the United States is limited and no standard processing method exists for the detection of HEV in foods. In order to develop a processing method for the detection of HEV in pork products, ground pork and pork liver were selected for method development. Murine norovirus (MNV) was used as a process control. A filtration step prior to RNA detection was shown to reduce the level of PCR inhibitors in ground pork and an additional ultracentrifugation process was successful in removing PCR inhibitors in pork liver. MNV RNA was detected in ground pork and liver samples inoculated with 4.7 log10 PFU/g and 3.0 log10 PFU/g, respectively. Using the developed method for viral RNA detection in ground pork and pork liver, 20 packages of ground pork (six 1 g sub-samples per package) and 14 pork livers (four 1 g sub-samples per liver) were screened for the presence of HEV RNA. Fifteen out of 119 (12.6%) ground pork samples tested positive for HEV RNA and 13 out of 20 packages (65%) contained at least one positive sample. Twenty-five of 56 (45%) of pork liver samples were positive for HEV RNA and 6 of 14 livers (43%) had all sub-samples test positive for HEV RNA. Overall, the results indicate ground pork and pig liver as a potential source of HEV.


Assuntos
Microbiologia de Alimentos/estatística & dados numéricos , Carne de Porco/virologia , Carne Vermelha/virologia , Animais , Hepatite E/epidemiologia , Vírus da Hepatite E/genética , Fígado/virologia , Produtos da Carne/virologia , Norovirus/genética , Prevalência , RNA Viral/análise , Suínos , Doenças dos Suínos/epidemiologia , Estados Unidos
10.
BMC Infect Dis ; 21(1): 7, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407198

RESUMO

BACKGROUND: Little is known about the etiology of childhood diarrhea in the United Arab Emirates (UAE) especially after the introduction of rotavirus vaccines. This study aimed to identify gastrointestinal pathogens in children with diarrhea (cases) and the carriage rate of these pathogens in asymptomatic children (controls). METHODS: Stool samples were collected from 203 cases and 73 controls who presented to two major hospitals in Al Ain city, UAE. Samples were analyzed with Allplex™ Gastrointestinal Full Panel Assay for common entero-pathogens. The association between diarrhea and the isolated pathogens was calculated in a multivariate logistic regression model. The adjusted attributable fractions (aAFs) were calculated for all pathogens significantly associated with cases. RESULTS: At least one pathogen was identified in 87 samples (42.8%) from cases and 17 (23.3%) from controls (P < 0.001). Rotavirus, norovirus GII and adenovirus were significantly more prevalent in cases. Their aAFs with 95% ci are 0.95 (0.64, 1.00) for rotavirus, 0.86 (0.38, 0.97) for norovirus GII and 0.84 (0.29, 0.96) for adenovirus. None of the 13 bacteria tested for were more commonly found in the cases than in controls. Cryptosporidium spp. were more significantly detected in cases than in controls. Co-infections occurred in 27.9% of the children. Viruses and parasites were significantly more likely to occur together only in the cases. CONCLUSIONS: Multiplex PCR revealed high positivity rates in both cases and controls which demand a cautious interpretation. Rotavirus remains the main childhood diarrhea pathogen in UAE. Effective strategies are needed to better control rotavirus and other causative pathogens.


Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Adenovírus Humanos/genética , Infecções por Caliciviridae/epidemiologia , Coinfecção/epidemiologia , Criptosporidiose/epidemiologia , Cryptosporidium/genética , Diarreia/epidemiologia , Norovirus/genética , Infecções por Rotavirus/epidemiologia , Rotavirus/genética , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/isolamento & purificação , Animais , Infecções por Caliciviridae/virologia , Estudos de Casos e Controles , Pré-Escolar , Coinfecção/parasitologia , Coinfecção/virologia , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Diarreia/parasitologia , Diarreia/virologia , Fezes/parasitologia , Fezes/virologia , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Norovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Infecções por Rotavirus/virologia , Vacinas contra Rotavirus , Emirados Árabes Unidos/epidemiologia
11.
Viruses ; 13(1)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33418922

RESUMO

Human noroviruses (HuNoVs) are one of the leading causes of foodborne illnesses globally. The viral genome is the most essential information for viral source tracing and viral transmission pattern monitoring. However, whole genome sequencing of HuNoVs is still challenging due to the sequence heterogeneity among different genotypes and low titer in samples. To address this need, in this study, the Transposase assisted RNA/DNA hybrid Co-tagmentation (TRACE-seq) method was established for next generation sequencing library preparation of HuNoVs. Our data demonstrated that almost the whole HuNoVs genome (>7 kb) could be obtained from all of the 11 clinical samples tested. Twelve genotypes including GI.3, GI.4, GI.5, GI.8, GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, and GII.21 were involved. Compared with the traditional method for viral metagenomics library preparation, optimized TRACE-seq greatly reduced the interference from the host's and bacterial RNAs. In addition, viral genome sequences can be assembled by using less raw data with sufficient depth along the whole genome. Therefore, for the high versatility and reliability, this method is promising for whole viral genome attainment. It is particularly applicable for the viruses with a low titer that are mixed with a complicated host background and are unable to be cultured in vitro, like the HuNoVs utilized in this study.


Assuntos
Biblioteca Gênica , Genoma Viral , Norovirus/genética , Norovirus/isolamento & purificação , Transposases/genética , Infecções por Caliciviridae/virologia , Doenças Transmitidas por Alimentos/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenômica , Hibridização de Ácido Nucleico/métodos , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA/métodos
12.
Int J Food Microbiol ; 337: 108931, 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33188986

RESUMO

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD95 was 102 genome copies/g for HAV, HEV and norovirus GII and 103 genome copies/g for norovirus GI. The LOQ was 2.90 × 104, 1.40 × 103, 1.60 × 104 and 1.30 × 104 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.


Assuntos
Microbiologia de Alimentos/métodos , Vírus da Hepatite A/genética , Vírus da Hepatite E/genética , Norovirus/genética , Surtos de Doenças/prevenção & controle , Água Potável/virologia , Frutas/virologia , Vírus da Hepatite A/fisiologia , Limite de Detecção , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Verduras/virologia
13.
J Formos Med Assoc ; 120(1 Pt 1): 212-216, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32448707

RESUMO

BACKGROUND/PURPOSE: The FUT2 gene is a histo-blood group antigen (HBGA) that determines the susceptibility to Norovirus (NoV) infection. This study investigated the clinical significance of the FUT2 gene profile and HBGA expression in NoV infection. METHODS: Fecal specimens were collected from children in Chang-Gung Children's Hospital with acute gastroenteritis (AGE). The medical records were reviewed for clinical data. The viral etiology of gastroenteritis was validated using molecular methods. Genomic DNA was isolated from saliva or whole blood with the Puregene B Kit, according to the manufacturers' instructions. Single-nucleotide polymorphisms (SNPs) were determined by real-time PCR assays. RESULTS: FUT2 gene DNA was examined in 98 children with AGE. NoV was detected by RT-PCR in 44 patients (44.8%), while 54 (55.2%) had non-NoV AGE. Of the 44 NoV patients, 38 (86.3%) were secretors (no G428A mutation) and six (13.7%) were non-secretors (G428A mutation). Of the 54 non-NoV AGE patients, 28 (51.9%) were secretors and 20 (48.1%) were non-secretors. NoV-infected patients who were secretors had more frequent vomiting (P < 0.001), longer duration of diarrhea (P < 0.001), and greater overall disease severity score (P < 0.001) compared with non-secretors. Non-NoV infection secretor AGE patients had a longer duration of diarrhea (P < 0.001) than non-secretors. CONCLUSION: FUT2 secretor status affects NoV AGE in children. Secretor patients have prolonged diarrhea, more frequent vomiting, more severe disease, and greater infection transmissibility than non-secretors.


Assuntos
Gastroenterite , Doença Aguda , Criança , Fucosiltransferases , Gastroenterite/genética , Genótipo , Humanos , Norovirus/genética , Taiwan
14.
J Biomed Nanotechnol ; 16(6): 954-964, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33187590

RESUMO

Clostridium difficile infection (CDI) has become the main cause of diarrhea-related diseases in domestic (China) inpatients. High-sensitivity and high-specificity detection methods for CDI must be applied clinically for CDI supervisory control. In this paper, we introduce a detection method for C. difficile and Norovirus based on real-time PCR. We developed and optimized a primer-probe for Norovirus targets tcdA and tcdB with remarkably increased detection sensitivity. We then used this method in an integrated cassette, and found increased detection efficiency for Norovirus standards in the cassette compared to C. difficile samples. These results provide a basis for further exploration of automatic testing system design.


Assuntos
Toxinas Bacterianas , Enterocolite Pseudomembranosa , Norovirus , Proteínas de Bactérias , China , Humanos , Norovirus/genética
15.
Arch Virol ; 165(12): 2767-2776, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32949263

RESUMO

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.


Assuntos
Infecções por Caliciviridae/diagnóstico , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Caliciviridae/genética , Fezes/virologia , Humanos , Norovirus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/química , Sensibilidade e Especificidade
16.
Proc Natl Acad Sci U S A ; 117(38): 23782-23793, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907944

RESUMO

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2 An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.


Assuntos
Infecções por Caliciviridae , Interações Hospedeiro-Patógeno/imunologia , Interferons , Intestinos , Norovirus , Sistemas CRISPR-Cas , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Humanos , Interferons/genética , Interferons/metabolismo , Intestinos/imunologia , Intestinos/virologia , Modelos Biológicos , Norovirus/genética , Norovirus/imunologia , Norovirus/patogenicidade , Organoides/imunologia , Organoides/virologia , Análise de Sequência de RNA , Transcriptoma/genética , Replicação Viral
17.
Braz J Med Biol Res ; 53(11): e9529, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32965324

RESUMO

Norovirus (NoV) is the main cause of gastroenteritis outbreaks worldwide. Although NoV spreads mainly from person to person, it is estimated that a large proportion of NoV outbreaks are caused by foodborne transmission. Bivalve mollusks are one of the most important foods involved in NoV transmission to humans. Little is known about NoV prevalence in shellfish harvested and commercialized in Brazil. The aim of this study was to map, for the first time, the distribution of NoV contamination in oysters and mussels harvested and commercialized in the coast of Pernambuco state, northeast Brazil. A total of 380 mollusks (260 oysters and 120 mussels) were collected between February and August 2017 either directly from harvesting areas or obtained from beach vendors at 17 sites in Pernambuco. Samples were processed and tested for NoV contamination using a SYBR Green real-time PCR assay. All samples were negative for NoV GI or GII contamination, suggesting a low risk of NoV contamination from this food source during the study period. Additional surveys in different areas of the Brazilian coast are warranted to monitor the risk of NoV infection upon seafood consumption.


Assuntos
Norovirus , Animais , Brasil/epidemiologia , Contaminação de Alimentos/análise , Humanos , Norovirus/genética , Alimentos Marinhos , Frutos do Mar
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(8): 734-739, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32958131

RESUMO

Objective To prepare monoclonal antibodies (mAbs) against GII.4 norovirus P domain by multiple antigens in an immunization program. Methods BALB/c mice were immunized with the multiple GII.4 NoV P domain, namely 1996cluster (VA387), 2004cluster, 2006b cluster and 2010 cluster. The spleen cells from the immunized mice were fused with SP2/0 cells and the hybridoma cells were screened by ELISA. The supernatant of the mAbs was collected and purified by the limiting dilution assay. Its subtype was identified, and the specificity and neutralization were analyzed by indirect ELISA and HBGA blocking, respectively. Results We obtained thirteen hybridoma cell lines that stably secreted mAbs against GII.4 NoV P domain. Their titers reached above 10-4 after purification. The subtypes of the mAbs were identified as IgG1. Indirect ELISA showed that all the mAbs specifically bound to all GII.4 norovirus variants. Five mAbs specifically bound to GII.17, GII.3 and GII.6 variants. Three mAbs specifically bound to GII.2 variants and strongly blocked NoV P particle from binding to the histo-blood group antigen (HBGA) receptors. Conclusion The mAbs against GII.4 norovirus P domain have been obtained by combined antigens immunization program. Multi-antigen immunization can enhance immune response significantly and cross-react with other GII.4 norovirus variants. The findings provide a basis for further development of novel GII.4 norovirus vaccines and for the optimization of the immunization programs of combined multi-antigen vaccine candidates.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Infecções por Caliciviridae , Norovirus , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/genética , Norovirus/imunologia
19.
Int J Food Microbiol ; 333: 108785, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-32717668

RESUMO

Norovirus in oysters is a significant food safety risk. A recent ISO detection method allows for reliable and repeatable estimates of norovirus concentrations in pooled samples, but there is insufficient data to estimate a distribution of copies per animal from this. The spread of norovirus accumulated across individual oysters is useful for risk assessment models. Six sets of thirty individual Crassostrea gigas oysters were tested for norovirus concentration levels by reverse-transcription quantitative PCR (RT-qPCR): three from a commercial harvest site, and three post-depuration. Five sets had norovirus GII means above the limit of quantification (LOQ), and one below the LOQ, but above the limit of detection. No norovirus GI was detected in pooled tests, and individual oysters were not tested for norovirus GI. Depuration was shown to reduce the mean concentration of GII copies, but not to affect the shape of the distribution around the mean. Deconvoluting the uncertainty of the method, the coefficient of variation was stationary (0.45 ±â€¯0.2). The best fit distribution was either a lognormal distribution or a gamma. Multiplying these distributions by the weight of oyster digestive tissues gave an estimate for the count mean. This was used as the parameter λ in three compound Poisson distributions: Poisson-lognormal, Poisson-gamma, and Poisson-K. No model was found to fit better than the others, with advantages for each. All three could be used in future risk assessments. Preliminary validation of sampling uncertainty using repeated testing data from a previous study suggests that these results have predictive power.


Assuntos
Crassostrea/virologia , Norovirus/isolamento & purificação , Frutos do Mar/virologia , Carga Viral/métodos , Animais , Contaminação de Alimentos/análise , Inocuidade dos Alimentos , Norovirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco/métodos
20.
Mar Pollut Bull ; 157: 111315, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32658680

RESUMO

Noroviruses are the most common cause of gastroenteritis outbreaks in humans and bivalve shellfish consumption is a recognized route of infection. Our aim was to detect and characterize norovirus in bivalves from a coastal city of Brazil. Nucleic acid was extracted from the bivalve's digestive tissue concentrates using magnetic beads. From March 2018 to June 2019, 77 samples were screened using quantitative RT-PCR. Noroviruses were detected in 41.5%, with the GII being the most prevalent (37.7%). The highest viral load was 3.5 × 106 and 2.5 × 105 GC/g in oysters and mussels, respectively. PMA-treatment demonstrated that a large fraction of the detected norovirus corresponded to non-infectious particles. Genetic characterization showed the circulation of the GII.2[P16] and GII.4[P4] genotypes. Norovirus detection in bivalves reflects the anthropogenic impact on marine environment and serves as an early warning for the food-borne disease outbreaks resulting from the consumption of contaminated molluscs.


Assuntos
Bivalves , Norovirus/genética , Animais , Brasil , Genótipo , Humanos , RNA Viral , Frutos do Mar
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