Kannika Pombubpa

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. J. Med. Virol. 81:345–353, 2009.

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. J. Med. Virol. 82: 854–860, 2010.

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.

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.

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.