Human bocavirus genotypes 1, 2, and 3 circulating in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand, 2012-2018.

BACKGROUND: Global distribution of human bocavirus (HBoV) has been known to associate with viral gastroenteritis in pediatric population. This study was conducted in Chiang Mai, Thailand from 2012 to 2018 to investigate epidemiology and genotype distribution of HBoV in pediatric patients less than 5 years old hospitalized with diarrhea. METHODS: A total of 2727 fecal specimens were investigated for the presence of HBoV using nested-PCR targeting partial VP1 capsid region. The detected HBoV strains were further characterized by nucleotide sequencing and phylogenetic analysis. RESULTS: Detection rate of HBoV infection in pediatric patients with acute diarrhea was 5.2%. Three genotypes of HBoV were detected with the most predominance of HBoV1 (50.4%), followed by HBoV2 (42.5%), and HBoV3 (7.1%). The majority of HBoV positive cases were children of 1 to <2 years old (31.9%) with high detection rate of HBoV1 and HBoV2. HBoV infection occurred all year-round. Phylogenetic analysis revealed that majority of HBoV1 displayed the genetic relationship with HBoV1 strains reported previously from Asia whereas only a few were related to the strains from Europe, South America, and Middle East. The HBoV2 and HBoV3 were also mainly closely related to the strains reported from Asia and a few from South America and North Africa. CONCLUSIONS: This study highlights distribution of HBoV genotypes circulating in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand. Overall, three genotypes of HBoV were detected with equally high prevalence of HBoV1 and HBoV2 whereas HBoV3 was detected with much lower prevalence.

Molecular Detection and Characterization of Picobirnavirus in Environmental Water in Thailand.

BACKGROUND: Enteric viruses are responsible for waterborne and foodborne infections affecting a large number of people around the world. Picobirnavirus (PBV) is a highly versatile virus, detected in a wide range of hosts and has been reported to be associated with gastroenteritis in humans and animals. METHODS: Molecular screening of environmental water samples for PBV was performed over a period of two years from November 2016 to July 2018. The virus was detected by RT multiplex-PCR, nucleotide sequencing, and phylogenetic analysis. RESULTS: Out of 125 water samples, 1.6% (2 samples) tested positive for PBV. Nucleotide sequence analysis showed that both PBV strains detected in this study belonged to PBV genotype II and most closely related to the human PBV genotype II reference strains previously detected in China, the Netherlands, and the USA. CONCLUSIONS: This study reports the first detection of PBV genotype II in environmental water in Thailand. Our result highlights the need for better sanitation and disposal of waste water within this area.

Molecular surveillance and genetic analyses of bufavirus in environmental water in Thailand.

Bufavirus (BuV), a recent discovery virus of the family Parvoviridae, has been suggested to be a potential causative agent of diarrhea in humans. The prevalence of BuV has been demonstrated mostly in fecal specimens of diarrheic patients. Little is known about the presence of BuV in the environmental water. The aim of the present study was to conduct a surveillance for BuV in the environmental water in Thailand. A total of 125 water samples were collected during November 2016 and July 2018 from six different areas in Chiang Mai city, Thailand. Water samples were concentrated and extracted to obtain BuV genomic DNA. The BuV was screened by amplification of NS1 region using nested-PCR. The detected BuV was further characterized by amplification of VP2 region. The NS1 and VP2 genes were sequenced and analyzed phylogenetically. The BuV strain (CMW88/18) detected in this study belonged to BuV1 with the prevalence of 0.8%. The CMW88/18 strain was most closely related to human BuV1 strains reported previously worldwide suggesting contamination of BuV in the environmental water could be a potential source of infection in human.

EDIII-DENV3 nanospheres drive immature dendritic cells into a mature phenotype in an in vitro model.

Domain III of E protein of dengue virus (DENV) is a target for vaccine development. Unfortunately, this protein based platform has low general immunogenicity. To circumvent this problem, the use of an adjuvant-nanoparticle delivery system to facilitate immunogenicity of soluble DENV-EDIII protein was investigated. One of the key features of this delivery system is its ability to simultaneously deliver antigens and exert adjuvanticity on specialized immune cells. In this study, N-trimethyl chitosan (TMC) nanoparticles (NPs) were generated to be used as adjuvant and carrier for soluble E-domain III of dengue virus serotype 3 (sEDIII-D3). Using ionotropic gelation, purified sEDIII-D3 was encapsulated into TMC NPs to form EDIII-D3 TMC NPs. After optimization, EDIII-D3 TMC particles exhibited a loading efficiency of 81% and a loading capacity of 41%. The immunogenicity of EDIII-D3 TMC NPs was tested using monocyte-derived dendritic cells (MoDCs). It was found that EDIII-D3 TMC NPs were well taken up by MoDCs. In addition, EDIII-D3 TMC NP treated MoDCs significantly upregulated maturation markers (CD80, CD83, CD86 and HLA-DR) and induced secretion of various cytokines and chemokines (IFN-α, IL-1ß, IL-6, IL-2, IL-12p70, IFN-γ, IL-4, IL-10, IL-8, MCP-1, macrophage inflammatory protein-1ß, granulocyte-colony stimulating factor, granulocyte-macrophage colony-stimulating factor and IL-7). These results indicate that EDIII-D3 TMC NPs are potent immunogens, at least in vitro, with the ability to induce maturation of DCs and highlight the potential use of TMC NPs for enhancing immunogenicity of a non-replicating dengue vaccine.

Responses of primary human nasal epithelial cells to EDIII-DENV stimulation: the first step to intranasal dengue vaccination.

BACKGROUND: About half of the world's population are living in the endemic area of dengue viruses implying that a rapid-mass vaccination may be required. In addition, a major target of dengue vaccine are children, thus, a needle-free administration is more attractive. These problems may be overcome by the alternative route of vaccination such as topical, oral and intranasal vaccination. Here, we investigated the possibility to deliver a dengue immunogen intranasally, a painless route of vaccination. The tested immunogen was the domain III of dengue serotype-3 E protein (EDIII-D3) loaded into trimethyl chitosan nanoparticles (EDIII-D3 TMC NPs). The primary human nasal epithelial cells, HNEpCs, were used as an in vitro model for nasal responses. RESULTS: At tested concentrations, EDIII-D3 TMC NPs not only exerted no detectable toxicity toward HNEpC cultures but also efficiently delivered EDIII-D3 immunogens into HNEpCs. Moreover, HNEpCs quickly and strongly produced proinflammatory cytokines (IL-1ß, IL-6, TNF-α), type-I IFN, the growth factors (GM-CSF, IL-7), the chemokines (MCP-1, MIP-1ß, IL-8), Th1-related cytokines (IL-2, IL-12p70, IL-17, IFN-γ) and Th2-related cytokine (IL-4) in response to EDIII-D3 TMC NPs treatment. CONCLUSIONS: A potential mucosal delivery system for dengue immunogens was revealed and found to stimulate a strong local innate antiviral response which possibly leading to a systemic adaptive immunity.

A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea.

A novel multiplex polymerase chain reaction assay was developed to identify 10 viruses in a single tube. The assay was targeted to detect group A and C rotaviruses, adenovirus, norovirus GI, norovirus GII, sapovirus, astrovirus, Aichi virus, parechovirus, and enterovirus. A total of 235 stool samples were collected from infants and children with acute gastroenteritis in Kyoto, Japan, from 2008 to 2009, then tested by this novel multiplex PCR and compared with a multiplex PCR described previously, which used 3 primer sets. The novel multiplex PCR could detect the targeted viruses in 111 of the 235 (47.2%) stool samples. Of these, 9 out of 10 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, sapovirus, adenovirus, parechovirus, group C rotavirus, astrovirus, and norovirus GI. In contrast, the multiplex PCR that used 3 sets of primers could detect the targeted viruses in 109 of the 235 (46.4%) stool samples. Among these, 8 types of viruses were identified, including group A rotavirus, norovirus GII, enterovirus, adenovirus, parechovirus, group C rotavirus, sapovirus, and astrovirus. The results suggested that the new multiplex PCR is useful as a rapid and cost effective diagnostic tool for the detection of major pathogenic viruses causing diarrhea.