Prevalence and molecular characterization of G1P[8] human rotaviruses possessing DS-1-like VP6, NSP4, and NSP5/6 in Japan.

During a survey of human rotaviruses in Okayama Prefecture, Japan in the 2011-2012 rotavirus season (between September 2011 and August 2012), G3P[8] was found to be a predominant genotype overall. However, G1P[8] emerged in the latter half of the season. To clarify the genetic background of the G1P[8] strains, the VP7, VP4, VP6, NSP4, and NSP5/6 genes of the strains were sequenced and genotyped. As a result, it was demonstrated that the strains with two different genotype constellations (G1-P[8]-I1-E1-H1 and G1-P[8]-I2-E2-H2) prevailed in the season. The G1P[8] strains possessing the DS-1-like VP6, NSP4, and NSP5/6 genes (the DS-1-like G1P[8] strains), which should reveal a short electropherotype, were originated from possible intergenogroup reassortment events. The DS-1-like G1P[8] strains accounted for 74.1% of all G1P[8] strains and were detected continuously throughout the season but not in the preceding season, indicating the possibility of new introduction and rapid spreading of these strains in the 2011-2012 season. The results suggest that the intergenogroup reassortants, considered generally unstable, can spread rapidly and become relevant.

A 4-year-old girl with clinically mild encephalopathy with a reversible splenial lesion associated with rotavirus infection.

Rotavirus is a common cause of severe gastroenteritis in children. It is known that rotavirus gastroenteritis may be accompanied by neurological manifestations, including encephalitis/encephalopathy and seizures. We report a case of a 4-year-old girl with clinically mild encephalopathy with a reversible splenial lesion associated with rotavirus infection. She was admitted to our hospital because of reduced level of consciousness, seizures, diarrhea, and vomiting. Fecal rotavirus antigen testing was positive. Cell counts in the cerebrospinal fluid (CSF) were normal. She had a normal serum sodium level on admission. Brain computed tomography showed no cerebral edema. However, electroencephalography showed generalized high-voltage slow waves, and diffusion-weighted magnetic resonance imaging demonstrated a transient abnormality in the splenium of the corpus callosum. We diagnosed clinically mild encephalopathy with a reversible splenial lesion associated with rotavirus infection. She recovered well and exhibited no neurological sequelae. Rotavirus RNA and antigen were not detected in the CSF, suggesting that the reversible splenial change was caused by indirect effects on the central nervous system subsequent to viral infection. Her normal serum sodium level indicates that this change can occur without hyponatremia.

Molecular characterization of OP354-like P[8] (P[8]b subtype) human rotaviruses A species isolated in Japan.

OP354-like P[8] (P[8]b subtype) species A rotaviruses (RVAs) were isolated first time in Japan during a RVA survey in Okayama Prefecture between 2006 and 2009. Two of 236 RVA-positive samples were identified as G1P[8]b by reverse transcription polymerase chain reaction. P[8]b strains (RVA/human-wt/JPN/OH1998/2008/G1P[8]b and RVA/human-wt/JPN/OH2024/2008/G1P[8]b) were isolated only in May, 2008 and both patients infected with P[8]b viruses lived in the same city, suggesting that the prevalence of P[8]b RVAs is limited considerably in Okayama Prefecture. Molecular analysis of four genes (VP4, VP6, VP7, and NSP4 genes) of Japanese P[8]b strains revealed that the VP4 genes of these strains were related closely to those of Southeast Asian and Indian P[8]b strains. In contrast, the VP6, VP7, and NSP4 genes of Japanese P[8]b strains were highly homologous to G1P[8]a strains prevalent in the same area. These results suggest that the Japanese P[8]b strain may be a result of reassortment events between Japanese G1P[8]a viruses and unidentified Asian viruses possessing the P[8]b VP4 gene.

Detection of group a rotavirus RNA and antigens in serum and cerebrospinal fluid from two children with clinically mild encephalopathy with a reversible splenial lesion.

We report on two children with mild encephalopathy with a reversible splenial lesion associated with group A rotavirus (GARV) infection. We examined stool, serum, and cerebrospinal fluid samples to determine the presence of the GARV VP7 gene and GARV antigen by reverse-transcription PCR and enzyme-linked immunosorbent assay, respectively. GARV antigen was detected in stool samples from both patients. The GARV G genotype was G9 in one child and G3 in the other. GARV antigens were also found in both serum samples. However, the GARV VP7 gene was detected in only one serum sample, which was collected on the first day of symptomatic illness. Neither GARV antigen nor the VP7 gene was detected in cerebrospinal fluid samples. Both patients had excellent outcomes. Our results suggest that the reversible splenial changes in our patients might have been caused by indirect effects to the central nervous system subsequent to viral infection.

Whole-genome characterization of human group C rotaviruses: identification of two lineages in the VP3 gene.

Group C rotavirus (GCRV) is distributed worldwide as an enteric pathogen in humans and animals. However, to date, whole-genome sequences are available only for a human strain (Bristol) and a porcine strain (Cowden). To investigate the genetic diversity of human GCRVs, nearly full-length sequences of all 11 RNA segments were determined for human GCRVs detected recently in India (v508), Bangladesh (BS347), China (Wu82 and YNR001) and Japan (OH567 and BK0830) and analysed phylogenetically with sequence data for GCRVs published previously. All the RNA segments of human GCRV strains except for the VP3 gene showed high levels of conservation (>93 % nucleotide sequence identity, >92 % amino acid sequence identity), belonging to a single genetic cluster distinct from those of animal GCRVs. In contrast, the VP3 genes of human GCRVs could be discriminated into two clusters, designated M2 and M3, that were distinguished phylogenetically from those of porcine and bovine GCRVs (clusters M1 and M4, respectively). Between M2 and M3, amino acid sequence identity of the VP3 gene was 84.1-84.7 %, whereas high identities were observed within each cluster (92.3-97.6 % for M2, 98.2-99.3 % for M3). Sequence divergence among the four VP3 clusters was observed throughout the amino acid sequence except for conserved motifs, including those possibly related to enzyme functions of VP3. The presence of obvious genetic diversity only in the VP3 gene among human GCRVs suggested that either the M2 or M3 VP3 gene of human GCRVs might have been derived through reassortment from an animal GCRV or from an unidentified human GCRV strain belonging to a novel genogroup.

Rotavirus antigenemia and genomia in children with rotavirus gastroenteritis.

We investigated group A rotavirus (GARV) antigenemia and genomia in children with rotavirus gastroenteritis. A total of 16 patients (2-29 months old), who received a diagnosis of GARV gastroenteritis using a commercial rapid test, were enrolled in this study. The sera from the patients were tested for the presence of GARV antigen and the VP7 and NSP3 genes using an enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction, respectively. Furthermore, when the VP7 gene was amplified, G type was identified and compared with that of GARV from the fecal samples of the patients. GARV antigen was detected in 12 of 16 serum samples (75.0%). No GARV antigen was found in infants that were 6 months old or younger. Thirteen of 16 serum samples (81.3%) were positive for GARV genes. In cases where both antigen and gene analyses were conducted, either GARV antigens or genes, or both, were detected in all cases. The GARV antigen levels of serum collected at 2 days of illness or more were significantly higher than were the levels in the samples obtained from the 1st day. Furthermore, the ELISA optical density values of patients with convulsion were significantly higher than were those of patients without convulsion, suggesting that the antigen level is associated with disease severity.

Detection of rotavirus RNA and antigens in serum and cerebrospinal fluid samples from diarrheic children with seizures.

Group A rotavirus (GARV) genes (the VP7 and NSP3 genes) in acute-phase cerebrospinal fluid (CSF), sera and stool samples from 6 children with convulsions accompanied by GARV gastroenteritis were investigated by reverse transcription-polymerase chain reaction (RT-PCR). When the VP7 gene was amplified from the samples, the G genotype (G type) of GARV was determined by RT-PCR. GARV genes were detected in the CSF samples of all 6 children, in 2 of the 3 blood samples, and in all of 4 stool samples. The G typing of GARV from 12 of a total of 13 samples indicated that G3 was the predominant G type in all samples. GARV antigens were detected by enzyme-linked immunosorbent assay in all of the 3 tested sera samples, while no GARV antigens were detected in any of the 5 tested CSF samples. We confirmed the presence of GARV genomes in the CSF samples from all of the children with rotavirus-associated seizures, including encephalopathy. However, the relationship between convulsions and the existence of GARV RNA in CSF remains unclear and further study is required.

Detection and molecular characterization of human group C rotaviruses in Okayama Prefecture, Japan, between 1986 and 2005.

A survey of human group C rotaviruses (CHRVs) was conducted in Okayama Prefecture, Japan, over a period of 19 years between 1986 and 2005. The presence of CHRVs was screened by enzyme-linked immunosorbent assay using CHRV-specific monoclonal antibodies and confirmed by reverse transcription-PCR. Of the 3,722 fecal specimens from sporadic cases of gastroenteritis, 44 specimens (1.2%) were positive for CHRV. The CHRV isolates were detected periodically but continuously, and the rates of positivity changed from one rotavirus season to the next. Moreover, the isolates were mainly detected in April and May, and the mean age of the patients infected with CHRV was 5.27 years. The genome electropherotypes (E types) of the isolates were classified into three patterns, and the dominant pattern changed from year to year. Nucleotide sequences of the VP7 and VP4 genes of 16 strains, which were representatives of 70 isolates from sporadic cases and outbreaks, were determined and analyzed. Although the VP7 and VP4 genes of the strains were closely related to each other, a phylogenetic analysis suggested that each of the VP7 and VP4 genes of the strains were grouped into three genetic lineages. Moreover, the strains could be divided into five types based on the combination of the E type and the genetic lineages of the VP7 and VP4 genes. These results indicate that CHRVs generally exist in Okayama Prefecture and that CHRVs with various genomic backgrounds prevailed in a limited area.

A nationwide epidemic of influenza C virus infection in Japan in 2004.

During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

Epidemiology of acute gastroenteritis outbreaks caused by Noroviruses in Okayama, Japan.

Noroviruses (called formerly "Norwalk-like viruses") cause food-borne gastroenteritis outbreaks. These outbreaks were thought to be related to shellfish consumption, although non-shellfish related outbreaks also occurred frequently in Japan. To clarify the epidemiology of Norovirus outbreaks, 435 stool samples were collected from 60 acute non-bacterial gastroenteritis outbreaks occurring over 8 years in Okayama, Japan. Using reverse transcription-PCR (RT-PCR), Noroviruses were detected in 257 cases (59.1% of all samples) from 46 outbreaks (77% of all outbreaks). The majority of the 46 Norovirus outbreaks (89%) occurred during November to March; notably one-third occurred in December. Restaurants, schools, and welfare institutions accounted for the major settings in 50%, 20%, and 15% of the Norovirus outbreaks, respectively. This was similar to other reports from Japan, but differed from those from the United Kingdom. The transmission routes were assigned in 27 of the Norovirus outbreaks. In 18 outbreaks the routes were related to human contact (7 from food handlers and 11 from person-to-person contact), whereas those related directly to shellfish occurred only in 9 outbreaks. These results suggest that transmission routes related to human contact are more important than recognized previously in the context of preventive medicine. Furthermore, all outbreaks in which some of the samples contained dual genogroups of Noroviruses were related to shellfish, suggesting that consumption of contaminated shellfish frequently results in mixed Norovirus infections in contrast to other transmission routes and that coexistence of genogroups is a useful marker for shellfish-related outbreaks.

[Outbreak of acute gastroenteritis caused by human group C rotavirus in a youth educational center in Okayama Prefecture].

In May 2000, an outbreak of acute gastroenteritis caused by human group C rotavirus (CHRV) occurred in a youth educational center located in the southern area of Okayama Prefecture. A total of 172 schoolchildren and teachers, who consisted of 51 persons belonging to F school and 121 persons belonging to K school, joined in an educational program at the center from May 24 to 26. Eighty-seven individuals (50.6%) of them showed clinical symptoms of gastroenteritis from May 24 to 30, and the outbreak peaked on May 27. The major clinical symptoms were abdominal pain (87.4%), diarrhea (50.6%), nausea or vomiting (21.8%), fever > 37 degrees C (12.6%), and headache (14.9%). The clinical symptoms of the patients in F school were more severe than those in K school. Thirty-two fecal specimens were collected from the patients and examined for gastroenteritis viruses by electron microscopy, ELISA, reverse passive hemagglutination test, and reverse transcription (RT)-PCR. As a result, CHRVs were detected in 21 specimens (65.6%) by RT-PCR. The possible route of the CHRV infection was thought to be a person to person transmission by following reasons: (i), CHRVs were detected in stools from patients who became ill on the first day of the program; (ii), CHRVs were not detected in stools from cooks; (iii), no possible causal food was found by epidemiological analysis of the outbreak. Furthermore, phylogenetic analysis of the VP7 gene among CHRVs isolated in Okayama revealed that the virus detected in this study was more closely related to the virus isolated from a sporadic case of gastroenteritis in 1996 than that isolated from an outbreak occurred in 1999.

Detection of human group C rotaviruses in Nigeria and sequence analysis of their genes encoding VP4, VP6, and VP7 proteins.

In a survey on the etiology of acute gastroenteritis in infants and young children in Nigeria, group C human rotaviruses were detected in two of 112 rotavirus positive stool specimens collected between 1999 and 2000. The VP7, VP6, and VP4 genes of the two Nigerian human group C rotavirus strains (Jajeri and Moduganari) were sequenced in this study. Comparative sequence analysis with other published human group C rotaviruses showed that the genes encoding the three structural proteins were remarkably conserved in primary structure with few mutations. The VP4 and VP7 genes from the two Nigerian strains were related more closely to each other than to those of other published strains, and formed a separate cluster on the phylogenetic tree. In contrast, it was of note that VP6 gene of strain Moduganari was related more closely to the Brazilian strain Belem than to the other Nigerian strain Jajeri. This is the first report of identification of human group C rotavirus in Nigeria and constitutes the first sequence data of human group C rotaviruses in the African continent.