Marcelo Takahiro Mitui

Detection of Human Bocavirus in the Cerebrospinal Fluid of Children With Encephalitis

We report 4 children with encephalitis associated with human bocavirus (HBoV) 1 or 2. All children were severely underweight, and 2 died; 1 of them had a matching HBoV2 nucleotide sequence isolated from serum and bocavirus like particles in the cerebrospinal fluid that were observed with electron microscopy. No further pathogens were detected in the cerebrospinal fluid of these patients.

Co-dominance of G1 and emerging G3 rotaviruses in Hong Kong: A three-year surveillance in three major hospitals

BACKGROUND The World Health Organization recommends rotavirus vaccines be included in all national immunization programs as part of a strategy to control diarrhoeal diseases. Sentinel surveillance is advised to monitor impact post-vaccine introduction and to document changes in genotype distribution. OBJECTIVES To determine the molecular epidemiology of circulating rotaviruses in Hong Kong prior to implementation of universal rotavirus vaccination. STUDY DESIGN From December 2004 through December 2007, 830 rotavirus-positive stool samples from subjects admitted for acute diarrhea to three major hospitals in Hong Kong were examined. The electropherotypes, and the G and P genotypes of these rotaviruses were determined. Phylogenetic analysis of the VP7 gene was performed. RESULTS G3P[8] was the dominant genotype (46.1%), followed by G1P[8] (36.5%) and G9P[8] (9.2%). A total of 35 electropherotypes were identified. The G3 and G1 strains had high sequence similarities among themselves and were clustered with strains from Asia particularly mainland China. The G9 strains were clustered with the globally spreading strains. G12 and G4 were not found. The prevalence of rotavirus infection peaked in winter season when temperature was low, atmospheric pressure was high, relative humidity was low and rainfall was negligible. CONCLUSIONS Genotype G3 and G1 were the dominant rotaviruses circulating in Hong Kong between 2004 and 2007. Strains were mainly related with those from mainland China. Ongoing surveillance of circulating genotypes should continue in anticipation of universal rotavirus vaccine introduction.

Serial passage of a street rabies virus in mouse neuroblastoma cells resulted in attenuation: potential role of the additional N-glycosylation of a viral glycoprotein in the reduced pathogenicity of street rabies virus.

Street rabies viruses are field isolates known to be highly neurotropic. However, the viral elements related to their pathogenicity have yet to be identified at the nucleotide or amino acid level. Here, through 30 passages in mouse neuroblastoma NA cells, we have established an attenuated variant of street rabies virus strain 1088, originating from a rabid woodchuck followed by 2 passages in the brains of suckling mice. The variant, 1088-N30, was well adapted to NA cells and highly attenuated in adult mice after intramuscular (i.m.) but not intracerebral (i.c.) inoculations. 1088-N30 had seven nucleotide substitutions, and the R196S mutation of the G protein led to an additional N-glycosylation. Street viruses usually possess one or two N-glycosylation sites on the G protein, 1088 has two, while an additional N-glycosylation site is observed in laboratory-adapted strains. We also established a cloned variant 1088-N4#14 by limiting dilution. Apart from the R196S mutation, 1088-N4#14 possessed only one amino acid substitution in the P protein, which is found in several field isolates. 1088-N4#14 also efficiently replicated in NA cells and was attenuated in adult mice after i.m. inoculations, although it was more pathogenic than 1088-N30. The spread of 1088-N30 in the brain was highly restricted after i.m. inoculations, although the pattern of 1088-N4#14s spread was intermediate between that of the parental 1088 and 1088-N30. Meanwhile, both variants strongly induced humoral immune responses in mice compared to 1088. Our results indicate that the additional N-glycosylation is likely related to the reduced pathogenicity. Taken together, we propose that the number of N-glycosylation sites in the G protein is one of the determinants of the pathogenicity of street rabies viruses.

Human bocavirus in patients with encephalitis, Sri Lanka, 2009-2010

We identified human bocavirus (HBoV) DNA by PCR in cerebrospinal fluid from adults and children with encephalitis in Sri Lanka. HBoV types 1, 2, and 3 were identified among these cases. Phylogenetic analysis of HBoV1 strain sequences found no subclustering with strains previously identified among encephalitis cases in Bangladesh.

Molecular characterization of a human group C rotavirus detected first in Turkey.

The present study was done to find out the prevalence of group B and C rotavirus infections in children with diarrhea presented at two major hospitals in Ankara, Turkey. Group B rotavirus was not found in any samples. One of 122 samples was positive for group C rotavirus. Phylogenetic analysis of genes for nonstructural protein NSP4, and structural proteins VP4, VP6, and VP7 confirmed the human origin of this strain. Similar to other human group C rotaviruses, one N-glycosylation site was predicted at amino acid residue 67 on the VP7 of strain GUP188. The genes of strain GUP188 were closely related to those of human group C rotavirus strain from the UK (Bristol) for NSP4, China (208 and Wu82) for VP4 and VP6, and from Colombia (Javeriana) for VP7, indicating that the Turkish group C rotavirus was unique and can serve as an additional reference strain for the molecular epidemiology of group C rotaviruses.

Detection and molecular characterization of diarrhea causing viruses in single and mixed infections in children: A comparative study between Bangladesh and Turkey

The incidence and mortality caused by diarrhea differ among countries. The prevalence of different enteric viruses, their molecular characteristics, and infections with multiple viruses might affect the disease incidence and mortality caused by diarrhea. The objective of this study was to determine the distribution and molecular characteristics of enteric viruses in children with diarrhea in Turkey and Bangladesh. A total of 288 stool samples that were negative for group A rotavirus were collected from children aged <5 years with acute diarrhea who presented to hospitals in Turkey and Bangladesh. The samples were screened for human bocavirus (HBoV), astrovirus (HAstV), norovirus (NoV), and adenovirus (AdV). Phylogenetic analyses of the targeted virus genes were performed. In Turkey, viruses were detected in 87/150 samples (58%), which included 69 (79.3%) with single viruses and 18 (20.7%) with multiple viruses. AdV was the most common virus, followed by HBoV. In Bangladesh, viruses were detected in 123/138 samples (89.1%), which included 29 (23.6%) with single viruses and 94 (76.4%) with multiple viruses. NoV GII was the most common, followed by AdV. The dominant genotypes among the virus species were HBoV 2A, HAstV 1, NoV GI type 1, and AdV 40. For NoV GII, the Hunter variant of genotype 4 in Turkey and genotype 17 in Bangladesh were the most common among the sequenced strains. It was concluded that the distribution of the viruses associated with diarrhea in Turkish and Bangladeshi children was different. Enteric viruses and mixed infections were more prevalent in Bangladesh than in Turkey. J. Med. Virol. 86:1159–1168, 2014.

Complete Genome Sequences of Two Astrovirus MLB1 Strains from Bhutanese Children with Diarrhea

ABSTRACT In addition to the eight genotypes of classic human astroviruses, seven new genotypes have been reported from two novel clades, MLB and VA. However, the epidemiology of these highly diverse astroviruses remains largely unknown. We report here the complete genome sequences of two MLB1 strains from Bhutanese children with diarrhea.

Inaccurate identification of rotavirus genotype G9 as genotype G3 strains due to primer mismatch

Reverse transcription (RT)-PCR is now the standard method for typing group A rotaviruses (RVA) to monitor the circulating genotypes in a population. Selection of primers that can accurately type the circulating genotypes is crucial in the context of vaccine introduction and correctly interpreting the impact of vaccination on strain distribution. To our knowledge this study is the first report from Asia of misidentification of genotype G9 as G3 due to a primer-template mismatch. We tested two published G-genotype specific primers sets, designed by Gouvea and colleagues (Set A) and Iturriza‐Gomara and colleagues (Set B) on RVA from Hong Kong and Sri Lanka. Among 52 rotaviruses typed as G3 by set A primers, 36 (69.2%) were identified as G9 by nucleotide sequencing and set B primers. Moreover, of 300 rotaviruses tested, 28.3% were untypable by set A primers whereas only 12.3% were untypable by set B primers. Our findings reinforce the need to periodically monitor the primers used for RVA genotyping.

Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs

Using the principle of immunochromatography, we previously developed a method called RAPINA (Rapid Neutralizing Antibody detection test) that can measure the level of rabies virus -neutralizing antibody (VNA) in serum samples [Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods 2009;161:58-62]. RAPINA is faster, simpler, and easier to perform compared with a virus-neutralizing test or enzyme-linked immunosorbent assay (ELISA). The improved version of RAPINA has greater positive and negative predictive values corresponding to a VNA level of 0.5 IU/mL, as recommended by the World Health Organization and the World Organization for Animal Health. To verify the efficacy of this improved method, serum samples were collected from humans and dogs before and after immunization against rabies and were tested in Japan, Sri Lanka, and Thailand. The results were compared between RAPINA and the true VNA levels measured by the Rapid Fluorescent Focus Inhibition Test (RFFIT). The improved RAPINA accurately predicted seropositivity for 182 of 183 seropositive human samples as assessed by RFFIT (99.5%) and for 138 of 140 RFFIT-negative human samples (98.6%). In dog serum samples, the positive and negative predictive values were 99.7% (345/355) and 95.6% (174/182), respectively. RAPINA was also used to estimate VNA levels in a semiquantitative manner by using serial dilution of serum samples. Our results show that RAPINA is an easy and rapid method for measuring VNA levels before and after immunization with the rabies vaccine and does not need a high skill level or sophisticated equipment. RAPINA can be used to monitor the success of preexposure prophylaxis in at-risk persons, vaccine coverage, and animal control. It can also be used in laboratories with modest facilities and where a large number of samples are screened.

Dominance of Emerging G9 and G12 Genotypes and Polymorphism of VP7 and VP4 of Rotaviruses from Bhutanese Children with Severe Diarrhea Prior to the Introduction of Vaccine

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged <5 years in Bhutan. Stool samples were collected from February 2010 through January 2011 from children who attended two tertiary care hospitals in the capital Thimphu and the eastern regional headquarters, Mongar. The samples positive for rotavirus was mainly comprised genotype G1, followed by G12 and G9. The VP7 and VP4 genes of all genotypes clustered mainly with those of neighboring countries, thereby indicating that they shared common ancestral strains. The VP7 gene of Bhutanese G1 strains belonged to lineage 1c, which differed from the lineages of vaccine strains. Mutations were also identified in the VP7 gene of G1 strains, which may be responsible for neutralization escape strains. Furthermore, we found that lineage 4 of P[8] genotype differed antigenically from the vaccine strains, and mutations were identified in Bhutanese strains of lineage 3. The distribution of rotavirus genotypes varies among years, therefore further research is required to determine the distribution of rotavirus strain genotypes in Bhutan.