Molecular epidemiology of noroviruses detected in Vietnamese children with acute gastroenteritis from 2012 to 2015.

Noroviruses, an important cause of diarrhoea in humans, are genetically diverse. The recent norovirus seasons recorded the emergence of new recombinants of the capsid and polymerase genotypes, with a global dominance of GII.Pe_GII.4 Sydney 2012 and GII.P17_GII.17 in Asian countries. However, the number of papers reporting the distribution of both polymerase and capsid genotypes circulating among children is scarce, with none from Vietnam. This study described both the polymerase and capsid genotypes of noroviruses circulating in Vietnamese children using stool specimens obtained under the World Health Organization rotavirus surveillance programme from 2012 to 2015. Of 350 specimens tested, noroviruses were detected in 90 (28 %) of 319 inpatient specimens and in 9 (29 %) of 31 outpatient specimens. The polymerase and capsid genotype combinations of GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 were co-dominant (51 and 24 %, respectively), both of which were recombinants, contributing to a high proportion (87 %) of recombinants among circulating noroviruses. GII.4 variants evolved in the same fashion in Vietnam as in other countries, with amino acid substitutions in the putative variant-specific epitopes of the protruding domain. Unlike neighbouring countries where the predominance of GII.P17_GII.17 was reported, only one GII.P17_GII.17 strain was detected from an outpatient in 2015 in Vietnam. In conclusion, a substantial burden due to norovirus gastroenteritis hospitalizations among Vietnamese children was associated with circulating co-dominant GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 strains. Continued surveillance is necessary to monitor infection caused by GII.4 variants and that of GII.P17_GII.17 noroviruses in paediatric patients in Vietnam.

Whole-genome characterisation of G12P[6] rotavirus strains possessing two distinct genotype constellations co-circulating in Blantyre, Malawi, 2008.

Rotavirus A strains detected in diarrhoeal children commonly possess any one of the genotypes G1, G2, G3, G4, and G9, with a recent increase in G12 detection globally. G12P[6] strains possessing short RNA (DS-1-like) and long RNA (Wa-like) migration patterns accounted for 27 % of the strains circulating in Blantyre, Malawi, between 2007 and 2008. To understand how the G12P[6] strains with two distinct genetic backgrounds emerged in Malawi, we conducted whole-genome analysis of two long-RNA and two short-RNA strains. While the former had a typical Wa-like genotype constellation of G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1, the latter was found to have G12-P[6]-I2-R2-C2-M1-A2-N2-T2-E2-H2: a VP3 gene mono-reassortant on the DS-1-like backbone. Phylogenetic and Bayesian Markov chain Monte Carlo analyses showed that the short-RNA G12P[6] strains were generated around 2006 by reassortment between an African Wa-like G12P[6] strain donating three genes (the VP7, VP4, and VP3 genes) and a G2P[4] strain similar to the one circulating in Thailand or the United States of America that donated the remaining eight genes. On the other hand, the long-RNA strains were generated as a result of reassortment events within Wa-like G12 and non-G12 strains commonly circulating in Africa; only the VP4 gene was from a Malawian G8P[6] strain. In conclusion, this study uncovered the evolutionary pathways through which two distinct G12P[6] strains emerged in Malawi.

Abrupt emergence and predominance in Vietnam of rotavirus A strains possessing a bovine-like G8 on a DS-1-like background.

An apparently single rotavirus A strain possessing a genotype constellation of G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 abruptly emerged, caused diarrhoea in children requiring hospitalisation, and increased to reach 27 % of strains detected during the first half of 2015 in Vietnam.

Molecular characterisation of wild-type G1P[8] and G3P[8] rotaviruses isolated in Vietnam 2008 during a vaccine trial.

Rotavirus vaccines work better in developed countries than in developing countries, leading to the question of whether the circulating strains are different in these two settings. In 2008, a clinical trial of the pentavalent rotavirus vaccine was performed in Nha Trang, Vietnam, in which the efficacy was reported to be 64 %. Although samples were collected independently from the clinical trial, we examined faecal specimens from children hospitalised for rotavirus diarrhoea and found that G3P[8] and G1P[8] were co-dominant at the time of the clinical trial. The aim of this study was to explore whether they were divergent from the strains circulating in the developed countries where the vaccine efficacy is high. Two G3P[8] and two G1P[8] strains that were regarded as representatives based on their electropherotypes were selected for full-genome sequencing. The genotype constellation was G1/G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. All but the VP4 genes, one of which belonged to the emerging P[8]b genotype (OP354-like VP4), clustered into one or more lineages/alleles with the strains circulating in developed countries, with ≥97.5 % nucleotide sequence identity. Additionally, 10 G1 and 12 G3 VP7 sequences as well as 31 VP4 sequences were determined. No amino acid differences were observed between the Vietnamese strains and strains in the developed countries that were likely to have affected the neutralisation specificity of their VP7 and VP4. In conclusion, apart from prevalent P[8]b VP4, virtually no differences were observed between the predominant strains circulating in Vietnam at the time of the clinical trial and the strains in the developed countries; hence, the lower vaccine efficacy was more likely to be due to factors other than strain divergence.

Determination of cut-off cycle threshold values in routine RT-PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis.

Norovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT-PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29-22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT-PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease.

Molecular epidemiology of rotavirus in cats in the United Kingdom.

Rotaviruses are leading causes of gastroenteritis in the young of many species. Molecular epidemiological studies in children suggest that interspecies transmission contributes to rotavirus strain diversity in people. However, population-based studies of rotaviruses in animals are few. We investigated the prevalence, risk factors for infection, and genetic diversity of rotavirus A in a cross-sectional survey of cats housed within 25 rescue catteries across the United Kingdom. Morning litter tray fecal samples were collected during the winter and summer in 2012 from all pens containing kittens and a random sample of those housing adult cats. Group A rotavirus RNA was detected by real-time reverse transcription-PCR, and positive samples were G and P genotyped using nested VP4 and VP7 PCR assays. A total of 1,727 fecal samples were collected from 1,105 pens. Overall, the prevalence of rotavirus was 3.0% (95% confidence interval [CI], 1.2 to 4.9%). Thirteen out of 25 (52%; 95% CI, 31.3 to 72.2%) centers housed at least one rotavirus-positive cat. The prevalence of rotavirus was associated with season (odds ratio, 14.8 [95% CI, 1.1 to 200.4]; P = 0.04) but not age or diarrhea. It was higher during the summer (4.7%; 95% CI, 1.2 to 8.3%) than in winter (0.8%; 95% CI, 0.2 to 1.5%). Asymptomatic epidemics of infection were detected in two centers. G genotypes were characterized for 19 (33.3%) of the 57 rotavirus-positive samples and P genotypes for 36 (59.7%). Two rotavirus genotypes were identified, G3P[9] and G6P[9]. This is the first population-based study of rotavirus in cats and the first report of feline G6P[9], which questions the previous belief that G6P[9] in people is of bovine origin.

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Molecular evolution of the VP7 gene of Japanese G2 rotaviruses before vaccine introduction.

Changes in the prevalence of G2 rotavirus after vaccine introduction are an important issue. However, such changes in a given country should be interpreted in the global context over time. We determined 35 Japanese G2 sequences and compared them with 508 globally collected G2 sequences. The D96N substitution, a substitution known to be associated with an abrupt increase in G2 strains and antigenic changes, emerged in those strains that formed a nascent lineage outside of the currently predominant lineage (sublineage IVa). Further studies are warranted to monitor the potential of their global spread, since they also appeared in Europe and Australia.

Multiple-dose therapy with bovine colostrum confers significant protection against diarrhea in a mouse model of human rotavirus-induced gastrointestinal disease.

Rotavirus is the most important etiologic agent of severe gastroenteritis. Previously, we reported that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal unimmunized cows at 6 to 7d after parturition effectively prevented against human rotavirus (HRV)-induced severe gastroenteritis in vivo, when administered as a single dose 60 min before viral inoculation. In the present study, we examined the efficacy of multiple administrations of SCBLC at smaller dosages after viral inoculation in vivo. We demonstrate that multiple administrations within 24h after virus inoculation resulted in earlier recovery from diarrheal symptoms, in an administration frequency-dependent manner. Furthermore, we investigated whether isolated IgG anti-HRV activity in SCBLC was equivalent to that of IgG isolated from bovine mature milk as measured by in vitro activity assays. We found that IgG-containing fractions from SCBLC and mature milk exhibited approximately the same level of anti-HRV activity. We concluded that the SCBLC contains a high level of IgG against HRV-induced severe gastroenteritis, which will be possible to use in protective effects in immunocompromised hosts, such as children and the elderly. Multiple doses of SCBLC during the early stages of infection or lower dosage of SCBLC given as a single dose both resulted in relief of diarrheal symptoms.

Glucocorticoid-induced TNF receptor-triggered T cells are key modulators for survival/death of neural stem/progenitor cells induced by ischemic stroke.

Increasing evidences show that immune response affects the reparative mechanisms in injured brain. Recently, we have demonstrated that CD4(+)T cells serve as negative modulators in neurogenesis after stroke, but the mechanistic detail remains unclear. Glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR), a multifaceted regulator of immunity belonging to the TNF receptor superfamily, is expressed on activated CD4(+)T cells. Herein, we show, by using a murine model of cortical infarction, that GITR triggering on CD4(+)T cells increases poststroke inflammation and decreases the number of neural stem/progenitor cells induced by ischemia (iNSPCs). CD4(+)GITR(+)T cells were preferentially accumulated at the postischemic cortex, and mice treated with GITR-stimulating antibody augmented poststroke inflammatory responses with enhanced apoptosis of iNSPCs. In contrast, blocking the GITR-GITR ligand (GITRL) interaction by GITR-Fc fusion protein abrogated inflammation and suppressed apoptosis of iNSPCs. Moreover, GITR-stimulated T cells caused apoptosis of the iNSPCs, and administration of GITR-stimulated T cells to poststroke severe combined immunodeficient mice significantly reduced iNSPC number compared with that of non-stimulated T cells. These observations indicate that among the CD4(+)T cells, GITR(+)CD4(+)T cells are major deteriorating modulators of poststroke neurogenesis. This suggests that blockade of the GITR-GITRL interaction may be a novel immune-based therapy in stroke.

The occurrence of amino acid substitutions D96N and S242N in VP7 of emergent G2P[4] rotaviruses in Nepal in 2004-2005: a global and evolutionary perspective.

Rotavirus is the leading cause of severe diarrhea among children worldwide. Strains with G2P[4] have captured recent attention because of its abrupt increase or re-emergence in many locations in the world. In Nepal, G2P[4] strains were detected at a rate of 1% in 2003-2004, but increased to 33% in 2004-2005. Thus, the VP7 genes of 45 emergent G2 strains from Nepal were sequenced and analyzed together with a total of 339 G2VP7 sequences detected over the last 34 years that were compiled from the DNA database. We found that all Nepalese VP7 sequences had a substitution from aspartic acid to asparagine at residue 96 (D96N) that was the hallmark of the lineage termed sublineage IVa, which replaced virtually all globally circulating G2 strains during the last decade. Within sublineage IVa, further sublineages emerged, of which a sublineage termed IVa-3 was identified to have another amino acid substitution from serine to asparagine at 242 (S242N). This sublineage, to which all Nepalese sequences belonged, now became the most frequent G2 sequence globally. In conclusion, the G2VP7 gene evolved in a dynamic fashion such that new lineages emerged within the previously dominant lineage, one of which became subsequently dominant.

Molecular characterization of rotavirus strains circulating among children with acute gastroenteritis in Madagascar during 2004-2005.

A survey was undertaken of the etiology of acute gastroenteritis in children <16 years of age in Antananarivo, Madagascar, from May 2004 through May 2005. With use of electron microscopy of fecal specimens, 104 (36%) of 285 children were found to be infected with rotavirus. Rotavirus strain characterization was undertaken using enzyme-linked immunosorbent assay, electropherotyping, reverse-transcription polymerase chain reaction genotyping, and nucleotide sequencing. The predominant group A rotavirus strain types identified were P[4]G2 (62%) and P[8]G9 (23%). Nucleotide sequence analysis of the VP7 genes of selected Malagasy G2 and G9 strains demonstrated similarity with those of other recently identified African rotavirus strains belonging to the same genotype.

Detection of coronaviruses in children with acute gastroenteritis in Maddina, Saudi Arabia.

BACKGROUND: The role of coronaviruses in paediatric gastro-enteritis is not well defined. We investigated the detection rate and epidemiological features of infection with coronavirus in children receiving hospital care for acute gastro-enteritis in Maddina, Saudi Arabia. METHODS: Stool specimens were collected from children less than 5 years of age who were either hospitalised in Maddina or given oral rehydration therapy as outpatients between April 2004 and April 2005. Coronaviruses were detected by electron microscopy. RESULTS: Coronaviruses were detected in 63 (6%) of 984 children with acute gastro-enteritis and were more commonly detected in outpatients (47/423, 11%) than in inpatients (16/561, 3%). The median age (range) of children with coronavirus infection was 42 months (10-60). Coronaviruses were detected throughout the year with the highest detection rate at the end of the winter season. CONCLUSIONS: Coronaviruses were commonly identified in children with diarrhoea in Saudi Arabia. Their role in paediatric gastro-enteritis warrants further evaluation.

Norovirus infection among children with acute gastroenteritis in Recife, Brazil: disease severity is comparable to rotavirus gastroenteritis.

Norovirus has captured increasing attention as an agent of childhood diarrhoea. However, it is not known whether norovirus causes as severe diarrhoea as rotavirus, particularly among children in developing countries. In a 1-year study conducted between May 2004 and April 2005 in Recife, Brazil, norovirus was detected by ELISA in 34/233 (15%) diarrhoeal children less than 5 years of age. The severity of clinical illness, as indicated by the presence of dehydration, the requirement for hospitalization, and the duration of hospital stay, was similar between children with norovirus and rotavirus infection. These data underscore the importance of norovirus as a cause of severe diarrhoea in children.

Apparent extinction of non-G2 rotavirus strains from circulation in Recife, Brazil, after the introduction of rotavirus vaccine.

The introduction of a G1P[8] rotavirus vaccine in Recife, Brazil, caused a decrease in rotavirus detection from 27% (March-May, 2006) to 5.0% (March-May, 2007), with all strains becoming G2, against which less protection had been predicted.

Significant association between leukoaraiosis and metabolic syndrome in healthy subjects.

OBJECTIVE: To investigate the relationship between leukoaraiosis (LA), which has been considered as an intermediate substitute of ischemic brain damages, and metabolic syndrome (MetS), which attracts attention as a risk factor for cerebrovascular diseases, in healthy subjects derived from various age groups. METHODS: We studied 1,030 healthy persons at ages between 28 and 78 years (mean, 52.7 years) with no history of stroke who visited a health care facility for routine health checkups. MetS was defined using the criteria of the National Cholesterol Education Program Adult Treatment Panel III. LA was assessed using the rating scale of the Atherosclerosis Risk in Communities study on MRI. Logistic regression analysis was performed to examine associations between LA and MetS. RESULTS: A total of 296 (28.8%) subjects had LA on MRI. MetS was significantly associated with the presence of LA (adjusted OR, 3.33; 95% CI, 2.30, 4.84). The association was constant across grades of LA; the adjusted OR was 3.41 (95% CI, 2.30, 5.06) for minimal LA and 3.07 (95% CI, 1.75, 5.38) for LA combining mild, moderate, and severe grades. As for MetS components, elevated blood pressure (adjusted OR, 2.16; 95% CI, 1.57, 2.99), impaired fasting glucose (adjusted OR, 1.64; 95% CI, 1.13, 2.39), and hypertriglyceridemia (adjusted OR, 1.56; 95% CI, 1.08, 2.28) were independently associated with all grades of LA. CONCLUSIONS: Metabolic syndrome (MetS) was significantly associated with every grade of leukoaraiosis (LA), including the minimal LA. Impaired fasting glucose and hypertriglyceridemia were associated with LA independently of elevated blood pressure. MetS can play an important role in identifying healthy subjects who have an increased risk of LA.

A lack of significant association between the electropherotype or G-serotype of the infecting strain and disease severity of rotavirus gastroenteritis.

Despite many previous studies, the question has not been settled as to whether some human rotavirus strains are more virulent than others. Since disease severity is most clearly reflected by the hospitalization status of the infected children, we examined whether there was any difference in the distribution of dominant strains between inpatient and outpatient groups. The study population comprised 763 children with acute diarrhea who were treated at a general hospital in Honjo City, Akita, Japan, during 1986-1997. Rotaviruses from stool specimens were classified into 77 electropherotypes using polyacrylamide gel electrophoresis. A single dominant strain or two co-dominant strains circulated simultaneously with some infrequent strains in most rotavirus seasons. Over the 11 rotavirus seasons, there was no significant difference in the relative frequencies of 15 rotavirus strains between the inpatient and the outpatient groups when strains of rotavirus were defined by their electropherotypes in polyacrylamide gel electrophoresis. However, infection with one G1 strain that co-dominated with a G4 strain carrying an identical electropherotype except the VP7 gene resulted in a statistically significantly reduced risk of hospitalization. There was no significant difference in the relative frequencies of four major G-serotypes or long/short RNA pattern. We conclude that the virulence or disease-causing potential of human rotavirus is not substantially different in the majority of strains.

A high incidence of intussusception in Japan as studied in a sentinel hospital over a 25-year period (1978-2002).

The development of second-generation rotavirus vaccines requires knowledge of baseline incidence rates for intussusception in infants prior to vaccine introduction. To obtain such estimates we reviewed clinical records in a hospital that served as the major provider of paediatric beds in a local community in the northern part of Japan. During the 25-year period (1978-2002), there were 91 hospitalizations due to radiologically confirmed intussusception in children <5 years of age, of which 45% were <1 year of age. Assuming that all children with intussusception in the area had been admitted to this hospital, there were an average of 185 and 78 hospitalizations per 100000 person-years for children <1 year old and 5 years old respectively. There was period-to-period variability with no long-term secular trend in the incidence of intussusception. The incidence rate in Japan was among the highest thus far reported, providing further evidence of geographic variability.

Rotavirus antigenemia in children with encephalopathy accompanied by rotavirus gastroenteritis.

Rotavirus antigen was detected in acute phase sera from 5 of 8 children with rotavirus-associated encephalopathy, confirming antigenemia. However, antigen was not detected in cerebrospinal fluid, failing to provide added evidence of invasion to the brain.

Relationship between shunt-dependent hydrocephalus after subarachnoid haemorrhage and duration of cerebrospinal fluid drainage.

Subarachnoid haemorrhage (SAH) patients in Fisher group 3 have a high risk of vasospasm and chronic hydrocephalus. We have provided cisternal irrigation combined with a head-shaking method for preventing vasospasm in SAH patients. We investigated 76 SAH patients in Fisher group 3 who received cisternal irrigation with head-shaking to evaluate the relationship between the occurrence of hydrocephalus and various clinical factors, including duration of cerebrospinal fluid (CSF) drainage. Chronic hydrocephalus occurred in 25 patients (33%). The occurrence of hydrocephalus was associated with longer duration of CSF drainage (median, 13 days versus 9 days). By logistic regression analysis using significant factors, including age, preoperative neurological grade and Glasgow Outcome Scale, only the duration of drainage was independently associated with the occurrence of hydrocephalus (Odds ratio = 1.18 per day; 95% confidence interval, 1.02- 1.36). These results indicate that long duration of CSF drainage for preventing vasospasm may increase the occurrence of hydrocephalus.