Detection of diarrheal viruses circulating in adult patients in Thailand

A total of 332 fecal specimens collected during January-December 2008 from adult patients with diarrhea were screened for group A and C rotaviruses, noroviruses GI and GII, sapovirus, Aichi virus, human parechovirus, enterovirus, adenovirus and astrovirus by RT-multiplex PCR. The detection rate for diarrheal viruses was 4.2 %. Adenovirus and enterovirus were equally detected as the most predominant viruses, with prevalence of 1.2 %, followed by Aichi virus (0.9 %) and norovirus GII (0.6 %). Mixed infection with norovirus GII and human parechovirus was also detected (0.3 %). This study provides epidemiological data for a wide variety of diarrheal viruses circulating in adult patients with diarrhea in Chiang Mai, Thailand.     

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

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.     

Molecular characterization of rare G3P[9] rotavirus strains isolated from children hospitalized with acute gastroenteritis

In 2004, an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand detected two uncommon human rotavirus strains (CMH120/04 and CMH134/04) bearing AU-1-like G3P[9] genotypes in 1 year old children hospitalized with acute gastroenteritis. The CMH120/04 and CMH134/04 rotavirus strains were characterized by molecular analyses of their VP6, VP7, VP8*, and NSP4 gene segments as well as the determination of RNA patterns by polyacrylamide gel electrophoresis (PAGE). Analysis of the VP8* gene revealed a high level of amino acid sequence identities with those of P[9] rotavirus reference strains, ranging from 94.9% to 98.3%. The highest identities were shared with the human rotavirus AU-1 strain at 97.8% and 98.3% for CMH120/04 and CMH134/04 strains, respectively. Analysis of the VP7 gene sequence revealed the highest identities with G3 human rotavirus strain KC814 at 96.6% and 96.2% for CMH120/04 and CMH134/04 strains, respectively. Based on the analyses of VP7 and VP8* genes, CMH120/04 and CMH134/04 belonged to G3P[9] genotypes. In addition, analyses of VP6 and NSP4 sequences revealed a VP6 subgroup (SG) I, with NSP4 genetic group C specificities. Moreover, both strains displayed a long RNA electrophoretic pattern. The finding of uncommon G3P[9] rotaviruses in pediatric patients provided additional evidence of the genetic/antigenic diversities of human group A rotaviruses in the Chiang Mai area of Thailand.     

Multiple combinations of P[13]-like genotype with G3, G4, and G5 in porcine rotaviruses

Epidemiological surveillance of porcine rotavirus (PoRV) strains was carried out in Chiang Mai Province, Thailand, from 2002 to 2003, and eight rotavirus isolates could not be completely typed by PCR. Of these, six were G3 and one was G4 and displayed a P-nontypeable genotype, while another isolate was both G and P nontypeable. Analysis of a partial VP4 gene of all eight P-nontypeable strains revealed a high degree of amino acid sequence identities (94.7% to 100%), suggesting that they belonged to the same P genotype. Comparison of the amino acid sequences of two representative strains (namely, strains CMP178 and CMP213) with those of 27 other known P genotypes revealed a high degree of amino acid sequence identity with those of P[13] porcine rotavirus reference strains HP113 and HP140, which were recently isolated in India. However, amino acid sequence comparison with non-P[13] rotavirus strains revealed relatively low identities, ranging from 58.2% to 84.8% for full-length VP4 sequences and 35.1% to 80.6% for VP8* sequences. Phylogenetic analysis revealed that CMP178 and CMP213 clustered together in a monophyletic branch with P[13]-like genotypes HP113 and HP140 which was clearly separated from the other lineages of P[13] or P[22] strains. Altogether, these findings indicate that PoRV strains CMP178 and CMP213 should be considered the P[13]-like VP4 genotype, a rare genotype that has been identified only in pigs. This study provides additional evidence of increasing genetic diversity among group A rotaviruses in nature.     

Detection and genetic characterization of cosavirus in a pediatric patient with diarrhea

Human cosavirus (HCoSV) is a newly discovered virus of the family Picornaviridae. A total of 411 fecal specimens were collected from children admitted to hospitals with acute gastroenteritis symptoms in Chiang Mai, Thailand, in 2010-2011 and screened for HCoSV by an RT-nested PCR method. HCoSV was found in a single specimen (CMH-N199-11) collected from a 3-year-old boy. This represents the first report of HCoSV infection in a pediatric patient with diarrhea in Thailand. Analysis of the complete coding sequence revealed that this HCoSV was most similar to the Chinese HCoSV-A reference strain SH1, and belonged to genotype A6. The data imply that HCoSVs detected in Thailand and China share the same evolutionary ancestor. Our results emphasize the need for further research to understand the distribution, genetic diversity, and association of the HCoSV with acute gastroenteritis in humans.     

Cytotoxic activity of indole alkaloids from Alstonia macrophylla.

Thirteen indole alkaloids isolated from the root bark of Alstonia macrophylla and a semisynthetic bisindole O-acetylmacralstonine have been assessed for cytotoxic activity against two human lung cancer cell lines, MOR-P (adenocarcinoma) and COR-L23 (large cell carcinoma), using the SRB assay. Pronounced cytotoxic activity was exhibited by the bisindoles on both cell lines. This suggests that, in comparison with the corresponding monomeric indoles, at least part of both the ring systems present in the bisindoles is essential for cytotoxic activity. The potent alkaloids were further tested against a human normal cell line (breast fibroblasts) and other human cancer cell lines including StMI1 1a (melanoma), Caki-2 (renal cell carcinoma), MCF7 (breast adenocarcinoma), and LS174T (colon adenocarcinoma). The bisindoles O-acetylmacralstonine, villalstonine and macrocarpamine were found to possess pronounced activity against cancer cell lines with IC50 values in the range of 2-10 microM, with no discernible cell-type selectivity. However, O-acetylmacralstonine displayed discernibly less toxicity against the normal breast fibroblasts.     

Human adenovirus type 1 related to feline adenovirus: evidence of interspecies transmission

Adenovirus is recognized to be a significant global enteropathogen in association with sporadic cases as well as outbreaks of acute gastroenteritis in humans. Based on the genetic analysis, one adenovirus strain bearing feline adenovirus gene was detected in a fecal specimen collected from a 1-year old female child with acute gastroenteritis in Japan. The human adenovirus detected and feline adenovirus shared high identities (100% and 97%) at the amino acid levels of hexon and fiber genes, respectively, and they belonged to the same human Ad1 cluster (known as the prototype Adenoid 71). These findings suggest that the interspecies transmission of adenovirus between humans and felines might occur in nature. This report is noteworthy because it is the first, to the best of our knowledge, providing evidence of adenovirus type 1 transmission between humans and animals, and highlights possible zoonoses in humans. Further epidemiological studies should be conducted to determine whether this adenovirus strain will be emergent in future.