Expression, purification and activity verification of human norovirus NS6 protein / 中华实验和临床病毒学杂志

Objective@#To express the NS6 nonstructural protein of human norovirus (NoV) in Escherichia coli, and to detect its enzymatic activity after purification.@*Methods@#Human NoV NS6 gene was cloned into the prokaryotic expression vector pDE1 and then was transformed into E. coli BL21 for expression. NS6 protein was purified by Ni-NTA affinity chromatography and Superdex 200 pg column. The activity of NS6 protein was determined by digestion of fusion protein 15VP1-6P at 37 ℃.@*Results@#Human NoV NS6 protein was stably and highly expressed in E. coli. After purification, the expressed product reached a purity of more than 95%, and the relative molecular weight of NS6 protein was about 23×103 Da. NS6 protein could cleave the fusion protein containing rhinovirus 3C cleavage site.@*Conclusions@#The nonstructural protein NS6 of human NoV was successfully expressed in Escherichia coli, which laid a foundation for further study on the pathogenesis of human NoV.

Analysis of surveillance results of viral diarrhea among children under 5 years of age in Shandong province from 2012 to 2017 / 中华实验和临床病毒学杂志

Objective@#To analyze the epidemiological characteristics and pathogen types of viral diarrhea in children under 5 years of age in Shandong province, and provide reference for the prevention and control of viral diarrhea.@*Methods@#A total of 1 017 fecal samples were collected from all children aged 5 years and younger with diarrhea who were admitted to the sentinel hospital of Shandong province from 2012 to 2017 within 3 days. Rotavirus antigen was detected by using an ELISA method . Rotavirus G/P typing was performed by RT-PCR; Norovirus (GI and GII), Sapovirus, and Astrovirus were detected by multiplex RT-PCR, and adenovirus was detected by PCR.@*Results@#In the 1 017 fecal specimens, the overall positive rate was 51.62% (525/1017), and viral nucleic acids were detected in at least 421 samples, and mixed virus infection was found in 104 sampes. The mixed infection accounted for 10.23% (104/1017) of all infections. The positive detection rates of Rotavirus, Calicivirus, Adenovirus and Astrovirus were 34.22% (348/1017), 16.91% (172/1017), 2.56% (26/1017), and 9.64% (98/1017)). The total detection rate of diarrhea virus and the detection rate of Rotavirus were the highest at 12 to 17 months of age, which was 51.72% (105/203) and 20.20% (41/203), respectively. The positive rate of diarrhea in children aged 2 years and younger was 49.36% (502/1017), which was much higher than the positive rate of diarrhea in children over 2 years old (2.26% (23/1017)). The peak of viral diarrhea was found to occur between November and April of the following year. The genotype of rotavirus was dominated by G9 (82.76%), the P genotype was dominated by P[8] (80.46%), and the G/P combination was dominated by G9P[8] (83.87%). Norovirus was the main infection in the Calicivirus (87.21%).@*Conclusions@#From 2012 to 2017, viral diarrhea in children under 5 years of age in Shandong Province was mainly caused by Rotavirus infection, followed by Norovirus. The overall prevalence of viral diarrhea in Shandong was moderate in China, and autumn and winter were the main epidemic season for viral diarrhea.

Structure and function of human norovirus nonstructural proteins / 中华实验和临床病毒学杂志

Norovirus (NoV), a single stranded RNA virus, is the major causative agent of the global acute gastroenteritis in humans, and it has drawn more and more attention. Because of the lack of appropriate animal models and in vitro cell culture models, people have studied and understood more about the epidemiology and genetic variation of human viruses. The functional characteristics of the viral encoded protein and the pathogenesis of virus infection are poorly understood. In this paper, we summarize the progress in studies on characteristics of non-structural protein of norovirus, and hope that with the recent breakthrough in human cell culture model of human norovirus, it can be used as a tool for further research on the function of human norovirus non-structural protein.

Identification of human Norovirus (HNoV) in domestic pig stool samples / Identificación de Norovirus Humano (HNoV) en muestras de estiércol de cerdos domésticos / Identificação de Norovírus Humano (HNoV) em amostras de suínos domésticos

Identificación de Norovirus Humano (HNoV) en muestras de estiércol de cerdos domésticos. Objetivo: determinar la presencia de NoVs como posible agente zoonótico causal de diarrea aguda entre cerdos y humanos. Materiales y métodos: se recolectaron un total de 77 muestras diarreicas provenientes de niños menores de cinco años y de cerdos menores de dos meses de la población La Chamba en el Tolima, Colombia. Estas muestras fueron transportadas al Laboratorio de Virología de la Pontificia Universidad Javeriana en Bogotá, donde inicialmente se les realizó extracción con Trizol-reagent, siguiendo las instrucciones del fabricante. Una vez obtenido el RNA, el siguiente paso fue hacer la RT-PCR para obtener el producto de amplificacion esperado para NoVs de 213 pb. Finalmente, las muestras positivas obtenidas en la RT-PCR fueron secuenciadas y analizadas mediante métodos bioinformáticos. Resultados: se obtuvieron seis muestras positivas de diarrea de niños y una muestra positiva de diarrea de cerdos, las cuales se evidenciaron en una banda de 231 pb. Cinco de las seis muestras positivas en niños y la muestra positiva en cerdos fueron secuenciadas y analizadas. Conclusiones: dada la estrecha relación genética que se evidencia entre las secuencias del cerdo y el humano, este podría ser un indicio de que exista la posibilidad de un animal en común como reservorio para cepas de humano u otras cepas de animales...

Objective. To determine the presence of NoVs as a possible causal zoonotic agent of acute diarrhea in pigs and humans. Materials and methods. We collected a total of 77 samples from diarrheal children under 5 years and pigs under 2 months from La Chamba town in Tolima, Colombia. These samples were transported to the Laboratory of Virology of the Pontificia Universidad Javeriana in Bogotá, and extraction with Trizol-reagent was done following the manufacturer’s instructions. After obtaining the RNA, the next step was to perform RT-PCR for obtaining the expected amplification product of 213- bp NoVs. Finally, the positive samples obtained in the RT-PCR were sequenced and analyzed by bioinformatics methods. Results. Six positive diarrheic samples from children and a positive diarrheic sample from pigs were detected by a band of 231 bp. Five of the six positive samples in children and the positive pig sample were sequenced and analyzed. Conclusion. Given the close genetic relationship between pig and human sequences, this could be an indication of the potential existence of a common animal acting as a reservoir for human or other animal strains...

Identificação de Norovírus Humano (HNoV) em amostras de suínos domésticos. Objetivo. Determinar a presença de NoVs como possível agente zoonótico causal de diarréia aguda entre porcos e seres humanos. Materiais e métodos. Foram coletadas um total de 77 amostras de crianças diarréicas menores de cinco anos e porcos com menos de dois meses da população “La Chamba” Tolima-Colômbia. Estas amostras foram transportadas ao laboratório de virologia da Pontifícia Universidade Javeriana - Bogotá, onde foram inicialmente submetidas à extração com Trizol reagment e seguindo as instruções do fabricante, após a obtenção do RNA o próximo passo foi realizar a RT-PCR para obter o produto de amplificação esperado para NoVs de 213 bp. Finalmente as amostras positivas obtidas no RT-PCR foram seqüenciadas e analisadas por métodos de bioinformática. Resultados. Foram obtidas seis amostras positivas de diarréia nas crianças e uma amostra positiva de diarréia em suínos, as que foram representadas em uma banda de 231 pb. Cinco das seis amostras positivas em crianças e a amostra positiva em suínos foram seqüenciadas e analisadas. Conclusões. Dada a estreita relação genética que se manifesta entre as seqüências de suínos e humanos, isso poderia ser uma indicação de que existe a possibilidade de um animal comum como reservatório para o humano ou outras cepas de animais...

Protein Expression of the Human Norovirus Capsid Gene using the Baculovirus Expression System

Human norovirus (HuNoV) is the major etiological agent of nonbacterial gastroenteritis worldwide. However, due to the absence of a rapid and sensitive diagnostic system, detection and monitoring have been limited. The HuNoV genome is composed of three open reading frames (ORFs). And major capsid protein, ORF2, is designated as a viral protein 1 (VP1). In this study, the baculovirus expression system was used for expression of the HuNoV capsid protein, VP1. Recombinant baculoviruses can be used as potent tools in HuNoV studies.