Recovery and Quantification of Norovirus in Air Samples from Experimentally Produced Aerosols.

Norovirus is the leading cause of acute gastroenteritis in humans across all age groups worldwide. Norovirus-infected patients can produce aerosolized droplets which play a role in gastroenteritis transmission. The study aimed to assess bioaerosol sampling in combination with a virus concentrating procedure to facilitate molecular detection of norovirus genogroup (G) II from experimentally contaminated aerosols. Using a nebulizer within an experimental chamber, aerosols of norovirus GII were generated at known concentrations. Air samples were then collected in both 5 mL and 20 mL water using the SKC BioSampler at a flow rate of 12.5 L/min, 15 min. Subsequently, the virus in collected water was concentrated using speedVac centrifugation and quantified by RT-qPCR. The optimal distances between the nebulizer and the SKC BioSampler yielded high recoveries of the virus for both 5 and 20 mL collections. Following nebulization, norovirus GII RNA was detectable up to 120 min in 5 mL and up to 240 min in 20 mL collection. The concentrations of norovirus GII RNA recovered from air samples in the aerosol chamber ranged from 102 to 105 genome copies/mL, with average recoveries of 25 ± 12% for 5 mL and 22 ± 19% for 20 mL collections. These findings provide quantitative data on norovirus GII in aerosols and introduce a novel virus concentrating method for aerosol collection in water, thus enhancing surveillance of this virus.

Molecular characterization of human bocavirus in recycled water and sewage sludge in Thailand.

The study aimed to assess the presence and molecular characterization of human bocavirus (HBoV) in recycled water and sewage sludge samples in Thailand. One hundred and two recycled water and eighty-six sewage sludge samples collected from a wastewater treatment plant were tested for the presence of HBoV using nested PCR with broad-range primer pairs targeting the capsid proteins VP1 and VP2. HBoV DNA was detected in recycled water of 9/102 (8.8%) samples and sewage sludge of 27/86 (31.4%) samples. Based on DNA sequencing and phylogenetic analysis, the HBoV DNA sequences had 98.8-100.0% nucleotide identity to the sequences from HBoV reported globally. Thirty-five HBoV-positive samples were identified to genotypes as the predominant HBoV2; 26 followed by HBoV3; 8 and the rare HBoV4; 1 sample. Concerning recycled water, HBoV2 was detected in 3 (2.9%) and HBoV3 was detected in 5 (4.9%) of all samples. The sewage sludge samples were characterized as HBoV2 in 23 (26.7%), HBoV3 in 3 (3.5%) and HBoV4 in 1 (1.2%) of all samples. The frequency of HBoV detected in recycled water and sewage sludge samples significantly differed in sample type (p-value = 0.007). The findings of three HBoV genotypes in recycled water and sewage sludge emphasized the circulation of the virus in the environment and the potential source of transmission to the community.

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand.

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n = 6), GII.4 (n = 1), GII.17 (n = 3), and GII.unclassified (n = 3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83-1.85 × 104 genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00 × 101 genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

Rotavirus Surveillance in Tap Water, Recycled Water, and Sewage Sludge in Thailand: A Longitudinal Study, 2007-2018.

The objective of this study was to describe the epidemiological and molecular surveillance of rotaviruses in tap water, recycled water, and sewage sludge in Thailand from 2007 to 2018. Three hundred and seventy tap water, 202 recycled water, and 72 sewage sludge samples were collected and processed to detect the rotavirus VP7 gene using RT-nested PCR. Rotavirus G genotypes were identified by DNA sequencing and phylogenetic analysis. The frequency of rotavirus detection was 0.54% of the tap water samples, 30.2% of the recycled water samples, and 50.0% of the sewage sludge samples. During the 12-year surveillance, G1 was prevalent most years and constantly predominant in recycled water and sewage sludge. G2 was identified in a tap water sample and in recycled water samples. G3 and G9 were observed in both recycled water and sewage sludge samples. The uncommon G6 rotavirus strain was identified in one recycled water sample. The rotavirus VP4 gene was detected in rotavirus strains with an identified G genotype using RT-multiplex nested PCR. The unusual P[6] genotype was the most frequently detected, followed by mixed P[6]/[4] and P[4] genotypes. Phylogenetic analysis of both G and P genotypes showed a close genetic relationship with sequences of human rotavirus strains. The high nucleotide identity of the rotavirus strains found in this study to human rotavirus strains suggests that the rotaviruses are derived from human source. These results represent useful epidemiological and molecular information for evaluating rotavirus distribution in water for consumption and irrigation, and in biosolids for agricultural application.

Norovirus Monitoring in Oysters Using Two Different Extraction Methods.

Detection of noroviruses in bivalve shellfish is difficult because of the low concentration of norovirus and the presence of reverse transcription (RT)-PCR inhibitors. This study aimed to assess the presence of noroviruses in oysters extracted using a proteinase K extraction (ISO 15216 method) and an adsorption-elution method. Seventy oyster samples were extracted using the two extraction methods and evaluated using RT-nested PCR. The results showed norovirus detection rates at an equal frequency of 28.6%, of which a total of 48 (68.6%) samples had corresponding positive or negative results, while there were 22 (31.4%) samples with discrepant results. Norovirus genogroup (G)I, GII, and mixed GI and GII were detected in 20%, 4.3%, and 4.3% of samples, respectively, by the proteinase K extraction method, which comprised of GI.2, GI.5b, GI.6b, GII.4, and GII.17 genotypes. With the adsorption-elution method noroviruses were detected in 17.1%, 8.6%, and 2.9% of samples, respectively, which comprised of GI.2, GII.2, GII.4, and GII.17 genotypes. All norovirus-positive oyster samples were further estimated for genome copy number using RT-quantitative PCR. The oyster samples processed using the adsorption-elution method contained norovirus GI of 3.36 × 101-1.06 × 105 RNA copies/g of digestive tissues and GII of 1.29 × 103-1.62 × 104 RNA copies/g. Only GII (2.20 × 101 and 7.83 × 101 RNA copies/g) could be quantified in samples prepared using the proteinase K extraction method. The results demonstrate the different performance of the two sample-processing methods, and suggest the use of either extraction method in combination with RT-nested PCR for molecular surveillance of norovirus genotypes in oysters.

Distribution of Naturally Occurring Norovirus Genogroups I, II, and IV in Oyster Tissues.

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 102 RNA copies/g of digestive tissues, 2.62 × 102 RNA copies/g of gills, and 1.61 × 103 RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.

Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (103 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters, Mussels, and Cockles in Bangkok, Thailand.

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10(-2) genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10(2) copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.

A comparison of virus concentration methods for molecular detection and characterization of rotavirus in bivalve shellfish species.

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species.

Rotavirus infection in children and adults with acute gastroenteritis in Thailand.

and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients, of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rota- virus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P[8] (27%), followed by G2P[4] (7%), G3P[8] (1%), and G9P[8] (1%). The rare genotypes identified were G1P[4], G1P[6], G2P[6], G2P[8], and G3P[6]. Mixed infections mostly occurred in children, comprising G1P[4]/P[8], G1P[4]/P[6], G1P[6]/P[8], G1/G2P[4], G1/G3P[4], and G1/G3P[4]/P[8]. Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10(8)/ml and in adult patients was 7.32x10(6)/ ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity. Rotaviruses are the most important cause of severe diarrhea in infants

Genetic diversity of rotavirus strains circulating in environmental water and bivalve shellfish in Thailand.

Rotavirus is a common cause of acute diarrhea in young children worldwide. This study investigated the prevalence and molecular characterization of rotavirus in environmental water and oyster samples in Thailand. A total of 114 water samples and 110 oyster samples were collected and tested for group A rotavirus using RT-nested PCR. Rotavirus genotype was identified by phylogenetic analysis of the VP7 genetic sequences. Group A rotavirus was detected in 21 water samples (18.4%) and six oyster samples (5.4%). Twenty five rotavirus strains were successfully sequenced and classified into four genotypes; G1, G2, G3, and G9. Rotavirus G1 (three strains), G2 (three strains), and G9 (two strains) demonstrated the genetic sequences similar to human strains (90%-99% nucleotide identity), whereas G3 (17 strains) was closely related to animal strains (84%-98% nucleotide identity). G1 strains belonged to lineages I (sub-lineage c) and II. G2 strains belonged to lineage II. G9 strains belonged to lineages III (sub-lineage b) and IV. G3 strains belonged to lineages I, III (sub-lineage c), and IV with a predominance of lineage I. The present study provides important information on the rotavirus strains circulating in the environment.

Detection and genetic characterization of norovirus in environmental water samples in Thailand.

The aim of this study was to detect and characterize noroviruses (NoVs) in environmental water samples. One hundred and fourteen water samples were collected from a river and irrigation canals in central Thailand during 2006-2007. NoVs were detected by RT-nested PCR in 13% of the samples. The river samples (22%) contained NoVs at a higher frequency than the irrigation canal samples (4%). Among the 15 NoV-positive samples, 9 harbored genogroup (G) I, 2 samples with GII, and 4 samples with mixed GI and GII. DNA sequencing of PCR amplicons and phylogenetic analysis of partial capsid gene revealed that 5 samples were of genotype GI-2, 1 sample was GI-6, and 1 sample was a mix of GI-2 and GII-unclassified genotypes. NoVs in water samples quantified using quantitative RT-PCR were in the range of 4.91 x 10(2) -1.26 x 10(3) copies/ml for NoV GI and 3.51 x 10(3) copies/ml for NoV GII. This is the first study demonstrating the presence of NoV variants in water samples collected from a river and the adjacent canals of Thailand.

A single injection of 19 kda carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(19)) formulated with Montanide ISA and CpG ODN induces protective immune response in mice.

OBJECTIVE: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection. METHODS: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM. RESULTS: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia. CONCLUSION: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

Noroviruses in oysters from local markets and oyster farms in southern Thailand.

One hundred and eighteen oyster samples collected from local markets and oyster farms in southern Thailand were examined for noroviruses (NoVs) and bacterial indicators of fecal contamination (fecal coliforms and Escherichia coli). Using a virus concentration procedure followed by RT-nested PCR, NoVs were detected in 38% of the samples. Oysters collected from oyster farms were found with NoVs at a higher detection rate (25/53 samples) than oysters from local markets (20/65 samples). Of the 45 NoV-positive oyster samples, 67% belonged to NoV genogroup I (GI), 15% to GII, and 18% to both GI and GII. DNA sequencing showed that 2 NoVs belonged to NoV GI-2 genotype. Fecal coliforms in NoV-positive oyster samples were in the range of < 3.0 to 1.5 x 10(4) most probable number (MPN)/g and 33% of NoV-positive oyster samples contained fecal coliforms within the standard acceptable level of raw shellfish (< 20 MPN/g). E. coli was found in the range of < 3.0 to 1.5 x 10(4) MPN/g and 9% of NoV-positive oyster samples were within acceptable levels of E. coli contamination (< 3 MPN/g). These findings indicate that NoV contamination in oysters obtained from both markets and oyster farms might pose a potential risk of acute gastroenteritis associated with raw oyster consumption. Examination for both fecal bacterial indicators and enteric viruses should be conducted for microbiological food safety of shellfish.

Detection of hepatitis A virus and bacterial contamination in raw oysters in Thailand.

This study was conducted to determine the presence of hepatitis A virus (HAV) in raw oysters (Crassostrea belcheri) using a virus concentration method and reverse transcription-nested polymerase chain reaction (RT-nested PCR). A total of 220 oyster samples were collected from oyster farms and local markets in Thailand. HAV was found in three oyster samples. Nested PCR products of HAV detected in oysters were characterized further by DNA sequencing of the VP1/2A region and subjected to phylogenetic analysis. All HAV sequences (168 basepairs) were associated with human HAV subgenotype IB (GIB). Fecal coliforms and Escherichia coli were determined using the multiple tube fermentation method, to assess the microbiological quality of collected oysters. Among oyster samples tested, 65% had fecal coliforms higher than the standard level for raw shellfish [< 20 Most Probable Numbers (MPN)/g]; MPN values in the range of 21.0-4.6 x 10(4)/g. Most oyster samples (85%) were contaminated with E. coli in the range of 3.0-4.6 x 10(4) MPN/g. One oyster sample with an acceptable level of fecal coliforms contained HAV GIB. E. coli was found in all HAV-positive oyster samples. The results suggest a significant presence of HAV and bacterial indicators of fecal contamination in raw oysters, which are a health risk for consumers and a source of gastrointestinal illness. Enteric viruses should also be tested to assess the microbiological quality of oysters.

Norovirus GII-4 2006b variant circulating in patients with acute gastroenteritis in Thailand during a 2006-2007 study.

Noroviruses (NoVs) are recognized as a significant cause of acute gastroenteritis in children and adults. A 14-month study, from January 2006 to February 2007, was undertaken in a hospital in Thailand to determine the prevalence and genetic characterization of NoVs in patients of all ages with acute gastroenteritis. Based on reverse transcription-nested polymerase chain reaction (RT-nested PCR), NoVs were detected in 122 of 273 (44.7%) collected stool samples. Of the 122 NoV-positive samples, 28 (23%) belonged to GI, 79 (64.8%) belonged to GII, and 15 (12.2%) were mixed infections of GI and GII strains. Three NoV GI-positive and 42 NoV GII-positive samples were characterized successfully by DNA sequencing of the RT-nested PCR products and phylogenetic analysis. For NoV GI, two genotypes were identified: GI-2 (one sample) and GI-6 (two samples). NoV GII could be classified further into five distinct genotypes: GII-2 (1 sample), GII-3 (3 samples), GII-4 (14 samples), GII-6 (3 samples), and GII-17 (2 samples), and one unclassified genotype (19 samples). All NoV GII-4 strains showed 88-98% nucleotide identity with NoV GII-4 2006b variants reported worldwide. Among genotypes of NoV characterized, one co-infected stool sample exhibited NoVs GI-6 and GII-4 2006b. This study suggests that there is an important role of NoVs as etiologic agents in patients with acute gastroenteritis. The predominant circulating genotype of NoV infections is GII-4 2006b variant.

Molecular characterization of rotaviruses, noroviruses, sapovirus, and adenoviruses in patients with acute gastroenteritis in Thailand.

Outbreaks of viral gastroenteritis occur worldwide including Thailand. Unfortunately, there is limited information since etiologic agents have not been identified in several outbreaks of nonbacterial gastroenteritis. The genotype of enteric viruses causing acute gastroenteritis in Thailand was determined using reverse transcription-multiplex polymerase chain reaction and DNA sequencing. From January 2006 to February 2007, stool samples were collected from patients with acute gastroenteritis of all age groups attending a hospital in Thailand, and patients with nonbacterial acute gastroenteritis (262 patients) were tested for enteric viruses. The overall positive detection rate of enteric viruses was 14.9%; group A rotaviruses (6.1%), noroviruses (6.5%): GI (0.8%) and GII (5.7%), adenoviruses (1.5%), and sapoviruses (0.8%) were found. Group B and C rotaviruses, and astroviruses were not detected in the enrolled patients. Viral acute gastroenteritis occurred in children less than 15 years of age (25.2%, 33/131) with higher frequency than in adults (4.6%, 6/131), P-value <0.001. Rotavirus G1 was the most predominant genotype, followed by G3, and G9. Among noroviruses, GI-2 was identified; whereas, GII was predominant with a high frequency of GII-4 observed, followed by GII-16, GII-2, GII-3, and GII-12. Sapovirus GII-3 and human adenoviruses were identified. This study suggests that enteric viruses play an essential role in patients with acute gastroenteritis attending hospital and mainly in children who have a higher prevalence of group A rotaviruses and noroviruses. The genetic analyses provide molecular epidemiological data for viruses important to public health.

Development of a method for concentrating and detecting rotavirus in oysters.

Identification of enteric viruses in outbreak-implicated bivalve shellfish is difficult because of low levels of contamination and natural inhibitors present in shellfish tissue. In this study, the acid adsorption-alkaline elution method developed in our laboratory was proposed for the detection of rotavirus from oyster samples. The acid adsorption-alkaline elution process included the following steps: acid adsorption at pH 4.8, elution with 2.9% tryptose phosphate broth containing 6% glycine, pH 9.0, two polyethylene glycol precipitations, chloroform extraction and reconcentration using speedVac centrifugation. Oyster concentrates were extracted for RNA and examined for rotavirus using reverse transcription-nested polymerase chain reaction (RT-nested PCR). A comparison of SuperScript One-Step RT-PCR system and RT followed by PCR before the nested PCR reaction showed the former detecting four-fold lower concentration of rotavirus (78.12 plaque forming units [PFU]/ml or 0.26 PFU/assay) than the latter (3.12 x 10(2) PFU/ml or 1.04 PFU/assay). In the seeding experiment, the developed acid adsorption-alkaline elution gave high sensitivity of rotavirus detection (125 PFU/g of oyster). From August 2005 to February 2006, 120 oyster samples (Crassostrea belcheri) were collected from local markets and oyster farms, concentrated, and tested for naturally occurring rotaviruses. Four oyster samples were group A rotavirus-positive. Based on phylogenetic analysis of rotavirus DNA sequences in those positive samples, the oyster samples contained the sequences associated with human rotavirus G9 (two samples), G3 (one sample), and G1 (one sample). The present study demonstrates the successful application of developed virus concentration method and RT-nested PCR for the detection of rotaviruses in naturally contaminated oyster samples. The method might be used as a tool for evaluating the presence of enteric viruses in shellfish for monitoring and control of public health.

The differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection.

BACKGROUND: Dengue haemorrhagic fever is an important public health problem and mainly occurs in children less than 15 years of age. Recently, the incidence of the disease have increased in adults but data on clinical and laboratory presentations of those affected are limited. OBJECTIVES: To assess and compare clinical manifestations and laboratory findings of dengue virus infected children and adults in Thailand. STUDY DESIGN: A 1-year study was conducted from September 2003 to August 2004 for dengue virus infected patients admitted to Phetchabun Provincial Hospital, Thailand. Physical signs, symptoms, and laboratory features were recorded. All dengue patients were confirmed using immunochromatographic test on convalescent sera. RESULTS: Based on serology-confirmed dengue virus infection, there was 286 dengue patients including 15 (5.3%) dengue fever and 271 (94.7%) dengue haemorrhagic fever (DHF). Among DHF cases, clinical classifications were DHF I, 40.9%; DHF II, 43%; and DHF III or dengue shock syndrome (DSS), 10.8%. Of all dengue patients, 231 cases (80.8%) were children aged less than 15 years and 55 cases (19.2%) were adults. The highest proportion of child cases was DHF I (42.9%), whereas that of adults was DHF II (51%). Some clinical manifestations were more common in adult patients, such as petechiae, melena, headache, retro-orbital pain, joint pain, myalgia, nausea and vomiting (p-value<0.05). Signs found commonly in children were epistaxis, oliguria, and liver enlargement (p-value<0.05). Haemoconcentration, thrombocytopenia, increased alanine aminotransferase, and longer prothrombin time were found to be significantly higher in adults than in children (p-value<0.05). CONCLUSIONS: Some clinical presentations of dengue disease and laboratory findings in adults are different from those in children. Therefore, adults as well as pediatric cases of DHF need appropriate and prompt case management to reduce the mortality rate of DHF.

Long-lasting protective immune response to the 19-kilodalton carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 in mice.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.