Genetic characterization of bulgarian rotavirus isolates and detection of rotavirus variants: challenges for the rotavirus vaccine program?

Annually 20-70% of all hospital admissions and 20% of fatal diarrhea cases among children less than 5 years of age occur due to severe rotavirus diarrhea. Universal immunization is the major strategy aimed at controlling rotavirus infection. The main objective of the present study was to elucidate the evolutionary relationships of the most common rotavirus strains co-circulating in Bulgaria. The sequence and phylogenetic analysis revealed strain diversity and circulation of different rotavirus variants belonging to a single genotype. A mutated G4P[8] strain with the insertion of an asparagine residue in position 76; G2, G9, and G1 variants with amino acid substitutions in the antigenic regions A, B, and/or C were all identified in this study in the absence of an immunization program. Rotavirus strain surveillance in both the pre- and post-vaccine eras is of increasing importance in order to assess the effectiveness of the rotavirus vaccines for protection against disease associated with a diverse population of rotavirus strains.     

Clinical and epidemiological characteristics of rotavirus infection in children of Cumaná, Venezuela

The detection rate of group A human rotavirus (HRV-A), as well as its association with clinical and epidemiological parameters, was studied in children younger than 5 years old with acute diarrhea attending to the University Hospital "Antonio Patricio de Alcalá" of Cumaná, between march 2006 and september 2007. Of 241 fecal samples collected in this study, 47 (19.5%) were positive to HRV-A by immunoassay. Rotavirus were present throughout the study and the major detection rates were on march, april and may of 2006 (rates were 30,0%, 28,6% y 43,8%, respectively) and september of 2007 (37,5%). Thirty four percent of cases with HRV-A occurred in children of 7 - 12 months and males were the most affected (55.3%), as well as the worker and marginal socioeconomic classes (72,4%). Children that not received maternal feeding were the group mainly infected by HRV-A (61.7%). Most of the children (72.4%) had one to four evacuations/day, with few vomits (38.2%) and fever (10.6%). Almost all the feces (83.0%) had a liquid or semi-liquid aspect. When these results were compared with previous data of the same geographic area, we observed a two-fold decrease of the detection rate of HRV-A and the clinical symptoms were the same as reported by other authors. Of 32 children vaccinated against rotavirus, 30 (93.8%) did not have HVR-A in their feces and there was a significant association between the vaccinated children and protection.     

Whole genome analyses of African G2, G8, G9, and G12 rotavirus strains using sequence-independent amplification and 454® pyrosequencing

High mortality rates caused by rotaviruses are associated with several strains such as G2, G8, G9, and G12 rotaviruses. Rotaviruses with G9 and G12 genotypes emerged worldwide in the past two decades. G2 and G8 rotaviruses are however also characterized frequently across Africa. To understand the genetic constellation of African G2, G8, G9, and G12 rotavirus strains and their possible origin, sequence‐independent cDNA synthesis, amplification, and 454^® pyrosequencing of the whole genomes of five human African rotavirus strains were performed. RotaC and phylogenetic analysis were used to assign and confirm the genotypes of the strains. Strains RVA/Human‐wt/MWI/1473/2001/G8P[4], RVA/Human‐wt/ZAF/3203WC/2009/G2P[4], RVA/Human‐wt/ZAF/3133WC/2009/G12P[4], RVA/Human‐wt/ZAF/3176WC/2009/G12P[6], and RVA/Human‐wt/ZAF/GR10924/1999/G9P[6] were assigned G8‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, G12‐P[4]‐I1‐R1‐C1‐M1‐A1‐N1‐T1‐E1‐H1, G12‐P[6]‐I1‐R1‐C1‐M1‐A1‐N1‐T1‐E1‐H1, and G9‐P[6]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2 genotypes, respectively. The detection of both Wa‐ and DS‐1‐like genotypes in strain RVA/Human‐wt/ZAF/3133WC/2009/G12P[4] and Wa‐like, DS‐1‐like and P[6] genotypes in strain RVA/Human‐wt/ZAF/GR10924/1999/G9P[6] implies that these two strains were generated through intergenogroup genome reassortment. The close similarity of the genome segments of strain RVA/Human‐wt/MWI/1473/2001/G8P[4] to artiodactyl‐like, human‐bovine reassortant strains and human rotavirus strains suggests that it originated from or shares a common origin with bovine strains. It is therefore possible that this strain might have emerged through interspecies genome reassortment between human and artiodactyl rotaviruses. This study illustrates the swift characterization of all the 11 rotavirus genome segments by using a single set of universal primers for cDNA synthesis followed by 454^® pyrosequencing and RotaC analysis. J. Med. Virol. 83:2018–2042, 2011. © 2011 Wiley‐Liss, Inc.     

Electropherotypes and serotypes of human rotavirus in Estonia in 1989–1992

Summary The electropherotypes and serotypes of human rotaviruses circulating in Tallinn (Estonia) in 1989–1992 have been studied. Rotaviruses were found in 372 (25.8%) of 1 442 faecal specimens of pediatric patients with acute diarrhea. Polyacrylamide gel electrophoresis of RNA from 318 isolates has revealed 41 electropherotypes. Frequent genomic alterations, including a shift of predominant electropherotypes, were observed during the study period. The serotype of 158 (50.3%) of 314 tested rotavirus isolates was identified using ELISA with VP7-specific monoclonal antibodies against 4 established human rotavirus serotypes. Serotype G1 was found to be largely predominant and accounted for 70.9% of the typeable specimens, serotypes G4, G2, and G3 accounted for 12%, 9.5%, 7.6%, respectively. A major shift to serotype G4 took place in 1990–1991. Serotype G1 was represented by the largest number of electropherotypes. All G1, G3 and G4 isolates were of long and all G2 isolates were of short electropherotypes. According to our results the isolates of an identical electropherotype belong to the same serotype.     

Characterization of rotavirus strains from hospitalized and outpatient children with acute diarrhoea in São Paulo, Brazil

From August 1994 to July 1995, 234 faecal samples from children with or without acute diarrhoea were collected and tested. The group of children with acute diarrhoea (A) was subdivided into two subgroups: subgroup A(1) was made up of children with severe diarrhoea, dehydrated and who needed hospitalization and subgroup A(2) was composed of children who only needed outpatient care. Group B was composed of children without acute diarrhoea (controls). Rotavirus was detected in 36.7% (18/49), 22.0% (15/68) and 1.7% (2/117) patients in groups A(1), A(2) and B, respectively. Of the 35 positive samples in which rotaviruses were detected the VP7 genotypes G1, G2, G3, G5 and the mixture (G2 + G5) were found in 40.0, 11.4, 11.4, 22.9 and 2.9% of the samples, respectively. Also, the VP4 genotypes P[8], P[4] and P[6] were detected in 57.1, 31.4 and 5.7%, respectively. Rotavirus VP6 subgroups I and II were detected at a frequency of 22.4 and 54.3%, respectively. Rotavirus RNA segments had short and long electrophoresis profiles in 20.0 and 51.4% of the cases, respectively. The severity of the disease was not related to a specific G and P types, subgroup or electropherotype.     

Molecular characterization of VP4 and NSP4 genes from rotavirus strains infecting neonates and young children in Belém, Brazil

Several reports have identified P[6] specificities in humans and in animals in different countries of the world, but few sequence data are available in public databases. In this work we have characterized the VP4 strains bearing P[6] specificity and NSP4 genotypes among diarrheic young children and diarrheic and non-diarrheic neonates from three studies previously conducted in Belém, Northern region of Brazil. As the to VP8* fragment, we observed a close relationship to both human prototypes of lineage P[6]-Ia (bootstrap of 99%) and porcine sublineages Ib and Ic (89.2-98.1% aa similarity and mean of 95%). With regards to the NSP4, the samples clustered into genotypes A and B. Of note, of the 27 P[6] strains analyzed in the present study and classified as genotype B, 8 (29.6%) were more similar to porcine prototypes when VP8* and NSP4 genes are compared, and were recovered, one from a neonate and seven from diarrheic children. These preliminary findings reinforce that further investigations are needed to assess the relative frequencies of P[6] strains in our region, as well as to investigate the potential for interspecies transmission involving humans and animals, particularly pigs.     

Evidence for zoonotic transmission of species A rotavirus from goat and cattle in nomadic herds in Morocco, 2012–2014

Virus Genes - Species A rotaviruses (RVAs) are a leading cause of diarrhea in children and in the young of a large variety of mammalian and avian host species. The purpose of this study was to...     

Diagnostic Accuracy and Visual Search Efficiency: Single 8 MP vs. Dual 5 MP Displays

This study compared a single 8 MP vs. dual 5 MP displays for diagnostic accuracy, reading time, number of times the readers zoomed/panned images, and visual search. Six radiologists viewed 60 mammographic cases, once on each display. A sub-set of 15 cases was viewed in a secondary study using eye-tracking. For viewing time, there was significant difference (F?=?13.901, p?=?0.0002), with 8 MP taking less time (62.04 vs. 68.99 s). There was no significant difference (F?=?0.254, p?=?0.6145) in zoom/pan use (1.94 vs. 1.89). Total number of fixations was significantly (F?=?4.073, p?=?0.0466) lower with 8 MP (134.47 vs. 154.29). Number of times readers scanned between images was significantly fewer (F?=?10.305, p?=?0.0018) with 8 MP (6.83 vs. 8.22). Time to first fixate lesion did not differ (F?=?0.126, p?=?0.7240). It did not take any longer to detect the lesion as a function of the display configuration. Total time spent on lesion did not differ (F?=?0.097, p?=?0.7567) (8.59 vs. 8.39). Overall, the single 8 MP display yielded the same diagnostic accuracy as the dual 5 MP displays. The lower resolution did not appear to influence the readers’ ability to detect and view the lesion details, as the eye-position study showed no differences in time to first fixate or total time on the lesions. Nor did the lower resolution result in significant differences in the amount of zooming and panning that the readers did while viewing the cases.     

Monitoring of group C rotavirus in children with acute gastroenteritis in Brazil: An emergent epidemiological issue after rotavirus vaccine?

Group C rotavirus (GpCRV) has a worldwide distribution; however, its epidemiology and ecology are still unclear. Evidence for a possible zoonotic role has been postulated recently for Brazilian children strains. The aim of this study was to monitor GpCRV in children ≤15 years with acute gastroenteritis during the 2007-2010 national Brazilian rotavirus surveillance, and to undertake the molecular characterization of the major VP6 capsid protein. A total of 3,019 fecal samples were first screened for Group A rotavirus (GpARV). A total of 2,205 GpARV ELISA negative samples were tested further for the presence of GpCRV by SDS-PAGE, electronic microscopy, and RT-PCR for the VP6 gene. The genetic diversity of GpCRV was carried out by sequencing the VP6 gene. GpARV and GpCRV infections were detected in 24.6% (742/3,019) and 0.3% (8/3,019), respectively. The GpCRV detection rate increased from 0.2% (1/422) in 2007 to 1% (7/708) in 2008, and GpCRV cases were not detected in 2009 and 2010. The phylogenetic analysis indicated that the strains belonged to the human lineage, and showed a genetic relationship with the GpCRV strain from Japan isolated in 2009. None of the study sequences was related closely to animal GpCRV strains. This study provides further evidence that GpCRV is a minor cause of acute childhood gastroenteritis in Brazil, and does not suggest that GpCRV may assume epidemiological importance in the future, even after the introduction of a GpARV vaccine. In addition, the molecular analyses of the GpCRV samples in this study do not support the zoonotic hypothesis.     

Predominance of G9P[8] rotavirus strains throughout France, 2014–2017

ObjectivesGroup A rotavirus is a major cause of acute gastroenteritis in young children worldwide. A prospective surveillance network has been set up in France to investigate rotavirus infections and to detect the emergence of potentially epidemic strains.MethodsFrom 2014 to 2017, rotavirus-positive stool samples were collected from 2394 children under 5 years old attending the paediatric emergency units of 13 large hospitals. Rotaviruses were genotyped by RT-PCR with regard to their outer capsid proteins VP4 and VP7.ResultsGenotyping of 2421 rotaviruses showed that after a marked increase in G9P[8] (32.1%) during the 2014–2015 season, G9P[8] became the predominant genotype during the 2015–2016 and 2016–2017 seasons with detection rates of 64.1% and 77.3%, respectively, whereas G1P[8] were detected at low rates of 16.8% and 6.6%, respectively. Phylogenetic analysis of the partial rotavirus VP7 and VP4 coding genes revealed that all of these G9P [8] strains belonged to the lineage III and the P [8]-3 lineage, respectively, and shared the same genetic background (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) as did most of previously detected G9P[8] strains and particularly the emerging G9P[8] strains from the 2004–2005 season in France.ConclusionsG9P[8] rotaviruses have become the predominant circulating genotype for the first time since their emergence a decade ago. In the absence of rotavirus immunization programmes in France, our data give an insight into the natural fluctuation of rotavirus genotypes in a non-vaccinated population and provide a base line for a better interpretation of data in European countries with routine rotavirus vaccination.     

套式PCR检测小儿咽拭中的MP,CT,UU

采用套式聚合酶链反应扩增技术检测小儿疑为呼吸道感染咽拭中的肺炎支原体（ＭＰ）、沙眼衣原体（ＣＴ）、解脲脲原体（ＵＵ）共１０７例，其中ＭＰ阳性２４例，阳性检出率为２２．４％，ＣＴ与ＵＵ未检出阳性，结果提示：小儿呼吸道感染可能以ＭＰ感染为主。     

Exclusive asymptomatic neonatal infections by human rotavirus strains having subgroup I specificity and “long” RNA electropherotype

Summary A large number of stool specimes, of healthy new born infants, collected from various hospitals and clinics in Bangalore City, India, have been examined for the presence of asymptomatic rotaviral excretion. Out of 370 samples analysed during a three year period from 1988 to 1991, 133 specimens (36%) were positive for rotavirus RNA. All these asymptomatic neonatal strains, without exception, showed long RNA pattern, but subgroup I specificity. Serotype analysis by ELISA or by hybridization with serotype-specific probes indicated that these strains probably represent a new serotype in newborn children. We find an exclusive association of human rotaviruses having long RNA pattern and subgroup I specificity with asymptomatic neonatal infections in contrast to the earlier observations of association of such unusual strains with acute gastroenteritis in young children.     

Are hospitalizations for rotavirus gastroenteritis associated with meteorologic factors?

Local climatic factors might explain seasonal patterns of rotavirus infections, but few models have been proposed to determine the effects of weather conditions on rotavirus activity. Here, we study the association of meteorologic factors with rotavirus activity, as determined by the number of children hospitalized for rotavirus gastroenteritis on the Mediterranean island of Mallorca (Spain). We conducted a retrospective review of the medical records of children aged 0–5 years admitted for rotavirus gastroenteritis between January 2000 and December 2010. The number of rotavirus hospitalizations was correlated to temperature, humidity, rainfall, atmospheric pressure, water vapor pressure, wind speed, and solar radiation using regression and time-series techniques. A total of 311 patients were hospitalized for rotavirus gastroenteritis in the 11-year study period, with a seasonal pattern from December to June, and a peak incidence in February. After multiple regressions, weekly rotavirus activity could be explained in 82 % of cases (p?2, respectively. In conclusion, hospitalization for rotavirus was strongly associated with mean temperature, but an association of rotavirus activity with solar radiation, atmospheric pressure, and wind speed was also demonstrated. This model predicted more than 80 % of rotavirus hospitalizations.     

Dissecting the Ca²⁺ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells

Rotavirus infection modifies Ca(2+) homeostasis provoking an increase in Ca(2+) permeation, cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), total Ca(2+) pools and, a decrease of Ca(2+) response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca(2+) homeostasis is not yet known. It has been proposed that the increase in [Ca(2+)](cyto) is the result of Ca(2+) release from intracellular stores, thereby activating store-operated Ca(2+) entry (SOCE). We studied the mechanisms involved in the changes of Ca(2+) permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca(2+) stores induced by thapsigargin or ATP was not able to elicit Ca(2+) entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca(2+)](cyto), is permeable to Mn(2+) and inhibited by La(3+) and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca(2+) entry is small and not modified by infection. The plasma membrane permeability to Ca(2+) in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca(2+) entry.     

Molecular analysis of VP4, VP7, and NSP4 genes of P[6]G2 rotavirus genotype strains recovered from neonates admitted to hospital in Belém, Brazil

This investigation describes the molecular characterization of P[6]G2 rotavirus strains from hospitalized neonates with community-acquired diarrhea (CAD), nosocomial diarrhea (ND), and asymptomatic nosocomial infection (ANI) in Belém, Brazil. Twenty-six rotavirus strains with P[6]G2 genotype were sequenced to genes coding for VP4, VP7, and NSP4 proteins. Phylogenetic analysis of the VP4 gene, including prototype strains RV3, ST3, M37, and U1205, showed that local P[6]G2 strains clustered forming a distinct lineage (bootstrap of 99%). Brazilian P[6]G2 strains had the highest homology (ranging from 96.0%-98.3%) with the African strain GR1107, G4P[6]. Phylogenetic tree for VP7 gene was constructed including old and new G2 African strains SA3958GR/97, SA356PT/96, SA514GR/87, SA4476PT/97, BF3676/99, GH1803/99, and representative strains of G1, G3, G4, G5, G8, and G9 genotypes. The Brazilian P[6]G2 samples fell into a distinct group (bootstrap value of 97%) and showed homology rates ranging from 92.1% to 93.5% with P[6]G2 African strains BF3676/99, GH1803/99, and SA3958GR/97. Nucleotide sequence analysis of the NSP4 gene, including human prototype strains S2, KUN, DS-1, RV5, RV3 and ST3, and animal prototype OSU, showed that all neonatal isolates fell into genotype A and clustered with a bootstrap value of 100%, with in-group similarities ranging from 99.3% to 100%. In this study no significant differences in nucleotide sequences of the VP4, VP7, and NSP4 genes could be observed when comparing diarrheic (CAD and ND) and non-diarrheic (ANI) babies. Monitoring of rotavirus strains in hospital environments is of particular importance, since it is claimed currently that an efficacious rotavirus vaccine, when available for routine use, will determine an impact on hospital-acquired rotavirus disease.     

Determination of the whole-genome consensus sequence of the prototype DS-1 rotavirus using sequence-independent genome amplification and 454® pyrosequencing

The prototype DS-1 rotavirus strain, is characterised by a short electropherotype and G2P[4] serotype specificity. Following sequence-independent genome amplification and 454^® pyrosequencing of genomic cDNA, differences between the newly determined consensus sequence and GenBank sequences were observed in 10 of the 11 genome segments. Only the consensus sequence of genome segment 1 was identical to sequences deposited in GenBank. A novel isoleucine at position 397 in a hydrophobic region of VP4 is described. An additional 7 N-terminal amino acids was found in NSP1. For genome segment 10 the first 34 and last 30 nucleotides of the 5′ and 3′-terminal ends, respectively, were identified. Genome segment 11 was found to be 821 bp long, which is 148 bp longer than the full length genome segment 11 sequence reported previously. This paper reports the first complete consensus genome sequence for the tissue culture adapted DS-1 strain free from cloning bias and the limitations of Sanger sequencing. Sequence differences in previous publications reporting on DS-1 rotavirus genome segment sequencing, were identified and discussed.     

Knowledge,motivations, expectations,and traits of an African,African-American,and Afro-Caribbean sequencing cohort and comparisons to the original ClinSeq® cohort

PurposeRacial minority populations are underrepresented in genomics research. This study enrolled African-descended individuals in a sequencing study and reported their characteristics.MethodsWe purposively recruited 467 individuals self-identified as African, African American, or Afro-Caribbean to the ClinSeq® study and surveyed them about knowledge, motivations, expectations, and traits. Summary statistics were calculated and compared with data from the study’s original cohort, which was primarily White and self-referred.ResultsRecruitment took five years and 83% of enrollees completed the survey. Participants had modest knowledge about benefits and limitations of sequencing (x̅s = 5.1, ranges: 0–10), and less than the original cohort (x̅= 7.5 and 7.7, respectively). Common motivations to enroll were learning information relevant to personal health (49%) or family members’ health (33%), and most had realistic expectations of sequencing. Like the original cohort, they had high levels of optimism, openness, and resilience.ConclusionEarly adopters may have relatively consistent personality traits irrespective of majority/minority status and recruitment methods, but high levels of genomics knowledge are not universal. Research should determine whether recruitment and consent procedures provide adequate education to promote informed choices and realistic expectations, which are vital to ethical research and increasing genomics research participation in underrepresented communities.     

Sensory coding, decoding, and representations. Unnecessary and troublesome constructs?

Reception of certain environmental energy patterns can allow organisms to successfully respond to present or future environmental conditions. Sensory systems are the means by which this reception occurs. The bioelectric components of this process are typically characterized as "sensory coding." "Coding" logically requires subsequent "decoding," and implies that the decoder has a special, commanding role. In contrast, the term "transformation," which is sometimes applied to the prebioelectric components of sensory systems, can connote a series of changes in the medium, form, or content of sensory processes. Neither coding nor decoding are implied, no notions of "representation" or "reconstruction" of the world are introduced, and distributed, parallel processing is a natural corollary of transformations. It is proposed that the problematic construct of sensory coding, with its concomitant decoding, be replaced with the more neutral and physical concept of transformations. Elimination of the notions of representation or reconstruction of the world in some special nervous system locus is also suggested.     

Gut microbiota, probiotics, and vitamin D: interrelated exposures influencing allergy, asthma, and obesity?

Current evidence supports a role for gut colonization in promoting and maintaining a balanced immune response in early life. An altered or less diverse gut microbiota composition has been associated with atopic diseases, obesity, or both. Moreover, certain gut microbial strains have been shown to inhibit or attenuate immune responses associated with chronic inflammation in experimental models. However, there has been no fully adequate longitudinal study of the relation between the neonatal gut microbiota and the development of allergic diseases (eg, atopic asthma) and obesity. The emergence of promising experimental studies has led to several clinical trials of probiotics (live bacteria given orally that allow for intestinal colonization) in human subjects. Probiotic trials thus far have failed to show a consistent preventive or therapeutic effect on asthma or obesity. Previous trials of probiotics have been limited by small sample size, short duration of follow-up, or lack of state-of-the art analyses of the gut microbiota. Finally, there is emerging evidence that the vitamin D pathway might be important in gut homeostasis and in signaling between the microbiota and the host. Given the complexity of the gut micriobiota, additional research is needed before we can confidently establish whether its manipulation in early life can prevent or treat asthma, obesity, or both.