多重RT-PCR用于临床检测三种胃肠炎病毒的研究

轮状病毒、诺瓦克病毒和星状病毒是引起病毒性胃肠炎的主要病原因子。研究采用JV12/JV13、P1/P2和Mon340/Mon348三对引物,建立了同时检测这3种病毒的多重RT-PCR技术,并应用于128份临床粪便样本的检测,检出轮状病毒62份(48.44%),诺瓦克病毒8份(6.25%),星状病毒11份(8.59%)。在灵敏度试验中,轮状病毒的检测灵敏度为5pg/mL、诺瓦克病毒和星状病毒的检测灵敏度均为50pg/mL。该研究所建立3种常见胃肠炎病毒的多重RT-PCR方法具有特异性强、灵敏度高的特点,可用于临床病原诊断和溯源。     

Molecular Detection and Characterization of Gastroenteritis Viruses Occurring Naturally in the Stream Waters of Manaus, Central Amazônia, Brazil

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.     

Assessment of gastroenteric viruses frequency in a children's day care center in Rio De Janeiro, Brazil: a fifteen year study (1994-2008)

This 15-year study aimed to determine the role of the main viruses responsible for acute infantile gastroenteritis cases in a day care center in the city of Rio de Janeiro, Brazil. From 1994 to 2008, 539 fecal samples were obtained from 23 outbreaks as well as sporadic cases that occurred in this period. The detection of Rotavirus group A (RVA), norovirus (NoV) and astrovirus (AstV) was investigated both by classical and molecular methods of viral detection. RVA was detected by enzymatic immune assay and/or polyacrylamide gel electrophoresis and genotyped by using semi-nested multiplex PCR. NoV and AstV were subsequently tested by real time PCR in all RVA-negative samples and genotyped throughout genome sequencing. Three protocols for molecular characterization of NoV nucleotide sequencing were performed with the partial nucleotide sequencing of genomic regions known as region B (polymerase gen), C and D (capsid gen).Viruses were identified in 47.7% (257/539) of the cases, and the detection rates of RVA, NoV and AstV in16.1% (87/539), 33.4% (151/452), and 6.3% (19/301), respectively. Most gastroenteritis cases were reported in autumn and winter, although NoV presented a broader monthly distribution. Viruses' detection rates were significantly higher among children aged less than 24 months old, although NoV cases were detected in all age groups. RVA genotypes as G1P[8], G9P[8], G2P[4], G3P[8] and G1+G3P[8] and RVA was no longer detected after 2005. NoV characterization revealed genotypes variability circulating in the period as GI.2, GI.3, GI.8 GII.2, GII.3, GII.4, GII.4 variants 2001 and 2006b, GII.6, GII.7, GII.12 and GII.17. AstV genotypes 1, 2, 4 and 5 were also characterized. Those data demonstrate the impact of NoV infection in cases of infantile gastroenteritis, surpassing RVA infection responsible for high morbidity rate in children under five years old.     

Continuous detection of norovirus and astrovirus in wastewater in a coastal city of China in 2014–2016

Norovirus (NoV) and human astrovirus (HAstV) are important causative agents of acute gastroenteritis in children and adults. They are comprised of multiple genotypes and attention should be paid to genotype changes or emergence of new genetic variants. To study the prevalence and diversity of NoV GI, GII, and HAstV circulating in eastern China, we conducted a three-year environmental surveillance in a coastal city of Yantai. Thirty-six sewage samples were collected, processed, and examined for the presence of viral genomes by PCR. The results showed that NoV GI, GII, and HAstV were detected in all 36 samples. Six NoV GI genotypes, 11 NoV GII genotypes, and 5 HAstV serotypes were identified; GI.6, GII.17, and HAstV-5 were the most prevalent types, respectively. Persistent existence of NoV GII.17 Kawasaki 308 variant was observed during whole study period. Phylogenetic analysis reflected multiple transmission lineages in local population for both viruses. Our results reflect continuous presence of enteric viruses in sewage, improve our understanding on their molecular epidemiology, and demonstrate surveillance on sewage is an effective approach in understanding the local circulation of enteric viruses.     

RNA viral community in human feces: prevalence of plant pathogenic viruses

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 10⁹ virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.     

Sequence analysis of the human virome in febrile and afebrile children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.     

Fruit consumption and seed dispersal of Dimorphandra mollis Benth. (Leguminosae) by the lowland tapir in the cerrado of Central Brazil.

Fruit phenology observations and consumption of Dimorphandra mollis (Leguminosae) were analyzed during seven months in an area of cerrado stricto sensu. We analysed 81 fecal samples collected at six different places of lowland tapirs (Tapirus terrestris) in central Brazilian cerrado. In addition, from the feces of five tapirs at the Brasília Zoo to which fruit had been offered, seeds were collected and used in germination tests. The results suggest that the tapir is an important fruit consumer and a potential seed disperser of D. mollis. In the field, however, fruit consumption was found to be very low, probably because of both fruit palatability and the low density of frugivores, especially tapirs. The possibility that the original dispersal agents of D. mollis seeds belonged to the South American Pleistocene megafauna is discussed.     

Rotavirus and adenovirus in Rondônia

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.     

Incidence of Norovirus and Other Viral Pathogens That Cause Acute Gastroenteritis (AGE) among Kaiser Permanente Member Populations in the United States, 2012–2013

Noroviruses and other viral pathogens are increasingly recognized as frequent causes of acute gastroenteritis (AGE). However, few laboratory-based data are available on the incidence of AGE caused by viral pathogens in the U.S. This study examined stool specimens submitted for routine clinical diagnostics from patients enrolled in Kaiser Permanente (KP) health plans in metro Portland, OR, and the Maryland, District of Columbia, and northern Virginia geographic areas to estimate the incidence of viral enteropathogens in these populations. Over a one-year study period, participating laboratories randomly selected stools submitted for routine clinical diagnostics for inclusion in the study along with accompanying demographic and clinical data. Selected stools were tested for norovirus, rotavirus, sapovirus, and astrovirus using standardized real-time RT-PCR protocols. Each KP site provided administrative data which were used in conjunction with previously published data on healthcare utilization to extrapolate pathogen detection rates into population-based incidence rates. A total of 1,099 specimens collected during August 2012 to September 2013 were included. Mean age of patients providing stool specimens was 46 years (range: 0–98 years). Noroviruses were the most common viral pathogen identified among patients with AGE (n = 63 specimens, 6% of specimens tested). In addition, 22 (2%) of specimens were positive for rotavirus; 19 (2%) were positive for sapovirus; and 7 (1%) were positive for astrovirus. Incidence of norovirus-associated outpatient visits was 5.6 per 1,000 person-years; incidence of norovirus disease in the community was estimated to be 69.5 per 1,000 person-years. Norovirus incidence was highest among children <5 years of age (outpatient incidence = 25.6 per 1,000 person-years; community incidence = 152.2 per 1,000 person-years), followed by older adults aged >65 years (outpatient incidence = 7.8 per 1,000 person-years; community incidence = 75.8 per 1,000 person-years). Outpatient incidence rates of rotavirus, sapovirus, and astrovirus were 2.0, 1.6, 0.6 per 1,000 person-years, respectively; community incidence rates for these viruses were 23.4, 22.5, and 8.5 per 1,000 person-years, respectively. This study provides the first age-group specific laboratory-based community and outpatient incidence rates for norovirus AGE in the U.S. Norovirus was the most frequently detected viral enteropathogen across the age spectrum with the highest rates of norovirus disease observed among young children and, to a lesser extent, the elderly. These data provide a better understanding of the norovirus disease burden in the United States, including variations within different age groups, which can help inform the development, targeting, and future impacts of interventions, including vaccines.     

Electron microscopy in the diagnosis of viral gastroenteritis in hospitalised children in the Czech Republic

Our study has been aimed at demonstrating the main role of viruses in the aetiology of acute gastroenteritis in children less than 5 years old and at pointing out the diagnostic potential of electron microscopy in the diagnosis of viral gastroenteritis. A prospective study was conducted to analyse the aetiology of diarrhoeal diseases in children less than 5 years of age admitted to the Department of Infectious Diseases between September 2006 and December 2008. All children were tested by faecal culture, latex agglutination and electron microscopy. A total of 832 children were included in the study. An aetiological agent was detected in 788 children (94.6 %). A bacterial aetiology was found in 22 (2.6 %) children and bacterial–viral co-infection was found in 146 (17.6 %) patients. The most frequent causative agents of gastroenteritis in children were viruses, which were detected in 620 (74.5 %) patients. The main causes of viral gastroenteritis were rotaviruses (detected in 410 children), followed by caliciviruses (42), coronaviruses (28), adenoviruses (19) and astroviruses (14). Dual viral infections were detected in 107 children, with rotavirus–calicivirus co-infection being the most common. Electron microscopy proved to be a more sensitive method in comparison with the latex agglutination test for the diagnosis of rotaviruses and adenoviruses. The major role of viruses in diarrhoeal diseases among children under 5 years of age in the Czech Republic has been confirmed. The diagnostic potential of electron microscopy, particularly in small outbreaks of gastroenteritis, was clearly shown.     

Prevalence and Genetic Diversity of Enteric Viruses in Children with Diarrhea in Ouagadougou,Burkina Faso

Enteric viruses are a major cause of diarrhea in children, especially those under five years old. Identifying the viral agents is critical to the development of effective preventive measures. This study aimed to determine the prevalence and genetic diversity of common enteric viruses in children under five years old in Burkina Faso. Stool samples from children with (n = 263) and without (n = 50) diarrhea disorders were collected in Ouagadougou, Burkina Faso from November 2011 to September 2012. Rotavirus, norovirus, sapovirus, astrovirus, adenovirus and Aichivirus A were detected using real-time or end-point (RT-)PCR. Rotavirus strains were G and P genotyped by multiplex RT-PCR and other viral strains were characterized by sequencing of viral subgenomic segements. At least one viral agent was detected in 85.6% and 72% of the symptomatic and asymptomatic patients, respectively. Rotavirus (63.5%), adenovirus (31.2%) and genogroup II norovirus (18.2%) were the most prevalent viruses in symptomatic patients, but only rotavirus and genogroup II norovirus were significantly associated with diarrhea (OR: 7.9, 95%CI: 3.7–17; OR: 3.5, 95%CI: 1–11.7, respectively). Sapovirus (10.3%), astrovirus (4.9%), genogroup I norovirus (2.7%) and Aichivirus A (0.8%) were less prevalent. The predominant genotype of rotavirus was G9P[8] (36.5%), and the predominant norovirus strain was GII.4 variant 2012 (71.4%). Among sapovirus, the genogroup II (87.5%) predominated. Astrovirus type 1 (41.7%) was the most frequent astrovirus identified. Aichivirus A belonged to the three genotypes (A, B and C). Enteric adenoviruses type 40 and 41 were identified in 10.2% and 5.1% respectively. Several cases of co-infections were detected. The results highlight the high prevalence and the high diversity of enteric viruses in Burkinabe children.     

人星状病毒研究进展

人星状病毒是引起婴幼儿病毒性腹泻的重要病原之一,本文回顾了其分子生物学特征、型别、所致疾病特征、致病机制、流行病学特征及检测方法等方面的文献,指出人星状病毒目前存在多种型别,尤其是近年来发现了多种新型的星状病毒,主要的流行型别是血清型1型,不仅引起肠道内感染,还可以引起其他系统疾病,所致疾病特征和其他腹泻相关病毒相似,但是其致病机制的研究尚不清楚,对于几种新发现的星状病毒,其与疾病的相关性研究较少,且未建立完善的实验室检测方法,还需要进一步的研究。     

Acute gastroenteritis and enteric viruses in hospitalised children in southern Brazil: aetiology,seasonality and clinical outcomes

Viral acute gastroenteritis (AG) is a significant cause of hospitalisation in children younger than five years. Group A rotavirus (RVA) is responsible for 30% of these cases. Following the introduction of RVA immunisation in Brazil in 2006, a decreased circulation of this virus has been observed. However, AG remains an important cause of hospitalisation of paediatric patients and only limited data are available regarding the role of other enteric viruses in these cases. We conducted a prospective study of paediatric patients hospitalised for AG. Stool samples were collected to investigate human adenovirus (HAdV), RVA, norovirus (NoV) and astrovirus (AstV). NoV typing was performed by nucleotide sequencing and phylogenetic analysis. From the 225 samples tested, 60 (26%) were positive for at least one viral agent. HAdV, NoV, RVA and AstV were detected in 16%, 8%, 6% and 0% of the samples, respectively. Mixed infections were found in nine patients: HAdV/RVA (5), HAdV/NoV (3) and HAdV/NoV/RVA (1). The frequency of fever and lymphocytosis was significantly higher in virus-infected patients. Phylogenetic analysis of NoV indicated that all of these viruses belonged to genotype GII.4. The significant frequency of these pathogens in patients with AG highlights the need to routinely implement laboratory investigations.     

我国河南与北京腹泻患儿中的星状病毒感染

人星状病毒（ＨＡｓｔＶ）是ＲＮＡ病毒的新家族,从我国河南和北京两地区的急性腹泻患儿便样中鉴定了７株星状病毒,经电镜观察病毒颗粒直径约为２８ｎｍ,表面呈五角或六角星状形态,用ＲＴ－ＰＣＲ检定为阳性,比较ＰＣＲ扩增产物的核苷酸序列构建的序列同源性树表明,１９９０年在河南流行的星状病毒主要为ＨＡｓｔＶ－１,另有一株ＨＡｓｔＶ－５,１９９６年北京流行株为ＨＡｓｔＶ－５。对ＨＡｓｔＶ患儿的临床症状进行了讨论     

Evidence of the co‐circulation of enteric viruses in sewage and in the population of Greater Cairo

Aims: To characterize major enteric viruses (enterovirus, rotavirus, norovirus, astrovirus and adenovirus) in the sewage of Greater Cairo and to compare the results with clinical data collected during the same period. Methods and Results: Seventy‐two sewage samples from two waste water treatment plants were collected from April 2006 through February 2007. Enteroviruses, noroviruses (NoVs) and rotaviruses (RVs) were detected by RT‐PCR in 22%, 18% and 8·3% of the samples, respectively. No adenovirus and astrovirus was detected. G2P[8], G9P[8], G1P[8], G2P[4] and rare G12 RV isolates were detected in the environment as well as a bovine RV. The environmental NoV strains mostly belonged to genogroup I (84%). Rotaviruses and some of the NoVs were similar to those found in the clinical samples at the same time. Conclusions: The comparison of environmental and clinical data suggests that similar RV and NoV isolates were circulating in the environment and in the population during the same period. Significance and Impact of the Study: Few studies have investigated the prevalence and the epidemiology of RVs and NoVs in Cairo. This work is the first to establish a correlation between viral gastroenteritis and the concomitant presence of enteric viruses in the environment for Greater Cairo where combined environmental and clinical surveys should help to prevent infections caused by these major pathogens.