Sequence analysis of the VP7 gene of human rotaviruses G2 and G4 isolated in Japan, China, Thailand, and Vietnam during 2001-2003.

Sequence and phylogenetic analyses of the rotavirus VP7 gene were performed on 52 human G2 and G4 strains isolated in Japan, China, Thailand, and Vietnam during 2001-2003. All genotype G2 strains included in the study clustered into lineage II of the phylogenetic tree, together with the majority of global G2 strains detected since 1995. The amino acid substitution at position 96 from aspartic acid to asparagine was noted among the emerging or re-emerging G2 rotavirus strains in Japan, Thailand, and Vietnam during 2002-2003. Genotype G4 strains detected in Vietnam grouped into lineage Ia of the phylogenetic tree, whereas Japanese G4 strains clustered in lineage Ic which included emerging G4 strains from Argentina, Italy, Paraguay, and Uruguay. It is noteworthy that an insertion of asparagine was found at position 76 in all the Japanese strains and that its presence might be involved in the emergence of G4 rotavirus in Japan during 2002-2003.

Dried umbilical cords in the retrospective diagnosis of congenital cytomegalovirus infection as a cause of developmental delays.

To clarify the impact of congenital cytomegalovirus (CMV) infection on developmental disabilities, 20 children with disabilities of unknown cause were analyzed. Five children were CMV positive and had no clinical manifestations at birth. Intracranial calcification was observed in 4 cases. Thus, congenital CMV infection is a significant cause of developmental disabilities.

Genetic linkage among human cytomegalovirus glycoprotein N (gN) and gO genes, with evidence for recombination from congenitally and post-natally infected Japanese infants.

Investigation of sequence polymorphisms in the glycoprotein N (gN; gp4273), gO (gp4274) and gH (gp4275) genes of human cytomegalovirus (HCMV) strains collected from 63 Japanese children revealed that their gO genotype distribution differed slightly from that of Caucasian populations and that there was a significant linkage between the gN and gO genotypes. Linkage of these genotypes in strains obtained from Caucasian populations has been reported, so our similar findings in Japanese infants are consistent with this, and suggest generality of this linkage. Sequence analysis suggests that recombination between two strains of different linkage groups occurred approximately 200 bp upstream of the 3'-end of the gO gene. Further studies are required to elucidate differences in biological characteristics among the linkage groups and the selective constraints that maintain the linkage.

Genetic variations in the gB, UL144 and UL149 genes of human cytomegalovirus strains collected from congenitally and postnatally infected Japanese children.

Human cytomegalovirus (CMV) is the leading cause of intrauterine viral infection. The association of genetic polymorphisms in some particular genes with the incidence and severity of congenital infection has been controversial. To address this issue, we analyzed the genotypes of the glycoprotein B (gB), UL144 and UL149 genes of CMV clinical strains obtained from 33 congenitally and 31 postnatally infected Japanese children. Our results demonstrated that (1) CMV strains with any combination of genotypes could be vertically transmitted from mother to fetus, potentially causing neurological abnormalities, (2) the gB3 genotype was more prevalent in the congenital cases than in postnatally infected children (P < 0.05), particularly in congenital cases with sensorineural hearing loss (P = 0.009), (3) there was no relationship between gB genotype and viral load in the urine and dried umbilical cord specimens in the congenital cases, and (4) the UL144 and UL149 genotype distributions had no bias for congenial infection. In future studies, it would be interesting to see whether the gB genotypes serve as a prognostic indicator of CMV-associated diseases.

Amino acid substitutions in the VP7 protein of human rotavirus G3 isolated in China, Russia, Thailand, and Vietnam during 2001-2004.

The distribution of rotavirus G-types in the world appears to be changing, especially with the emergence of G3 and G9 in many countries. Sequence analysis of the VP7 gene was performed on the 27 human G3 rotavirus strains isolated in China, Russia, Thailand, and Vietnam during 2001-2004. All the strains studied were clustered into the same branch of the phylogenetic tree. The comparison of the G3 deduced amino acid sequences between the studied Chinese strains and the strains circulating in China during 1986-1992 showed a wide range of amino acid substitutions (up to 13 amino acids in the VP7 antigenic regions). The two considerable changes both from aspartic acid to asparagine were located at positions 96 in antigenic region A and 213 in antigenic region C. Those amino acid substitutions of the Chinese G3 strains might involve in the emergence of G3 rotavirus in China during 2001-2003.

Sequence analysis of the VP7 gene of human rotavirus G1 isolated in Japan, China, Thailand, and Vietnam in the context of changing distribution of rotavirus G-types.

Over the last decade, rotavirus G1 has represented the most common genotype worldwide. Since 2000, the prevalence of rotavirus G1 has decreased in some countries such as Japan and China. To monitor the trend of the VP7 encoding gene of rotavirus G1, we performed a sequence analysis of 74 G1 rotavirus strains isolated in Japan, China, Thailand, and Vietnam during the period from 2002 to 2005. The phylogenetic tree showed that all of the studied G1 strains from the four countries clustered into lineage III, the same as the majority of the G1 strains isolated in China and Japan in 1990 and 1991. Examination of the deduced amino acid sequences of the G1 strains from China and Japan revealed an amino acid substitution at position 91 (Asn instead of Thr) in antigenic region A when compared to the G1 strains isolated in China and Japan in 1990, 1991, and global reference strains. For the G1 strains from Thailand and Vietnam, there were three amino acid substitutions, not belonging to any antigenic regions. The study showed that there have been no considerable changes of human rotavirus G1 isolated in Japan, China, Thailand, and Vietnam. Further studies need to be carried out for a better understanding of why such changes in the prevalence of rotavirus G1 occur in these countries.

Diversity of viruses associated with acute gastroenteritis in children hospitalized with diarrhea in Ho Chi Minh City, Vietnam.

A molecular epidemiological study on common diarrheal viruses was conducted in Ho Chi Minh City, Vietnam between October 2002 and September 2003. Fecal samples were collected from 1,010 hospitalized children with acute gastroenteritis. Those samples were screened for groups A, B, and C rotavirus, adenovirus, genogroups I and II norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) by RT-multiplex PCR, and the positive specimens were characterized further by ELISA, nested PCR, or sequencing. Among the diarrheal viruses detected, group A rotavirus was the most common, with a proportion of 67.4%, whereas NoV GII, adenovirus, SaV, and HAstV were also found in 5.5, 3.2, 0.8, and 0.6%, respectively. It is noteworthy that the group C rotavirus was first reported in Vietnam, with a proportion of 0.5% in this study. Fifty-six of 1,010 (5.5%) samples were found positive with more than one viral agent, in which 25 samples contained both group A rotavirus and NoV GII. Group A rotavirus could be identified throughout year with the peaks in both the dry and rainy season, whereas other viruses prevailed mainly in the rainy season. G-typing for the group A rotavirus showed that genotype 1 was still the most prevailing (33.0%), but interestingly, serotype 9 was emergent and became the third most common rotavirus G-type in these samples (13.7%). The four most common G-P combinations globally, G1P[8], G2P[4], G3P[8], and G4P[8] were found in 46.8% of rotavirus-positive samples, and it is of interest that one unusual rotavirus G9P[19] strain was first detected in Vietnam. The majority of NoV strains belonged to GII/4, and SaV strains mainly clustered with the Manchester strain (GI/1). Twenty-seven out of 32 adenovirus strains were identified as serotype 41. All HAstVs belonged to genotype 1. The results indicated clearly the impact of viral agents causing gastroenteritis and the importance of vaccination against diarrhea in Vietnam.

Novel intragenotype recombination in sapovirus.

Based on the genetic analysis, a novel, naturally occurring recombination between two distinct sapovirus subtypes (subtype a and subtype b) within genogroup I genotype 1 was identified. Breakpoint analysis of recombinant sapovirus showed that the recombination site was at the polymerase-capsid junction. This is the first report of the existence of acute gastroenteritis caused by intragenotype recombinant sapovirus. The results also provided evidence that the natural recombination occurs not only in sapovirus genogroup II but also in sapovirus genogroup I.

Development of a novel protocol for RT-multiplex PCR to detect diarrheal viruses among infants and children with acute gastroenteritis in Eastern Russia.

Viral gastroenteritis is one of the most common illnesses in humans worldwide and it has a great impact on people. Recently, we reported three RT-multiplex PCR assays termed A, B and C that could detect three groups of diarrheal viruses; group A, B and C rotaviruses and adenovirus [Phan et al., J Med Virol 2004; 74:173-9]; norovirus GI and GII, sapovirus and astrovirus [Yan et al., J Virol Meth 2003; 114:37-44]; enteroviruses, hepatitis A and E viruses and influenza A virus [Phan et al., Arch Virol 2005; 1175-85], respectively. In the present study, we developed a novel protocol with a small volume of reaction mixture for RT and PCR products (only 8 microl and 11 microl, respectively) that can amplify genomes of target viruses simultaneously. A total of 100 fecal specimens from infants and children with acute gastroenteritis in Birobiclzhan city, Eastern Russia, were collected during November 2003 and March 2004 and tested for the presence of those viruses by the novel RT-multiplex PCR protocol. Group A rotavirus was the most prevalent (67%), followed by norovirus GII (4%), group C rotavirus (1%), and sapovirus (1%). Interestingly, one fecal specimen turned out to be positive for hepatitis A virus. The sensitivity and specificity of RT-multiplex PCR assays with a novel protocol demonstrated a strong validation against the previously published RT-multiplex PCR. The findings clearly indicated that this novel protocol is simple and cost-effective to investigate the molecular epidemiology of acute gastroenteritis caused by diarrheal viruses. This report is the first, to our knowledge, detecting hepatitis A virus in feces from diarrheal infants and children in Eastern Russia.

Outbreak of acute gastroenteritis associated with group A rotavirus and genogroup I sapovirus among adults in a mental health care facility in Japan.

An outbreak of acute gastroenteritis consisting of 57 cases occurred in a mental health care facility in Takasaki city, Japan during 6th February and 27th March 2002. A total of 18 fecal specimens collected from 17 residents and one member of the medical staff during this outbreak were tested for the presence of viral enteropathogens by RT-PCR and latex agglutination. Group A rotavirus and sapovirus were detected in 5 out of 18 fecal specimens (55.6%). To our knowledge, this is the first finding of an outbreak of gastroenteritis associated with co-circulation of different kinds of viruses such as group A rotavirus and sapovirus. All of group A rotaviruses were typed further as P[4]G2 strains. Both rotavirus and sapovirus were subjected to molecular analysis by sequencing. It was noteworthy that all rotaviruses and sapoviruses had high homologies, respectively, to each other and sapoviruses presented a potential novel sapovirus genogroup I (GI) genotype, which was obviously different from any GI genotypes (GI-a, b, c, and d). The outbreak associated with these viruses spread gradually from dormitory to dormitory, suggesting a spread by person-to-person contact, although investigation on the route of transmission of the outbreak is lacking. The findings confirm the presence of group A rotavirus and sapovirus are important in acute gastroenteritis among adults in Japan.

Development of RT-multiplex PCR assay for detection of adenovirus and group A and C rotaviruses in diarrheal fecal specimens from children in China.

Rotavirus, adenovirus, norovirus, sapovirus and astrovirus are considered to be significant global enteropathogens associated with sporadic cases and outbreaks of acute gastroenteritis. Therefore, a rapid and sensitive assay is preferred to screen for the presence of these viruses in diarrheal fecal specimens. In a previous study, we developed a reverse transcription single-round multiplex polymerase chain reaction (RT-smPCR) assay for the simultaneous detection of norovirus (genogroup I, genogroup II), sapovirus and astrovirus in fecal specimens (Yan et aL, 2003). Recently, we developed another RT multiplex PCR for one-step amplification of all subgenera A to F adenoviruses, and group A and C rotaviruses. In this study, a total of 207 fecal specimens collected from children with acute gastroenteritis between December 2001 and April 2003 in Yunnan Province, China were examined for the presence of adenoviruses, and group A and C rotaviruses, by RT-multiplex PCR. The detection rate of these three viruses was 55.1% (114 out of 207 specimens), among which adenovirus and group A and C rotaviruses were identified in 11, 101 and 1 fecal specimen, respectively. Furthermore, one specimen was found to be positive for co-infection with adenovirus and group A rotavirus. An epidemic of acute gastroenteritis was also identified as peaking mainly in October and November. Taken together, our results clearly indicate that this novel assay provides a potentially rapid and convenient tool for epidemiologic investigation of diarrhea caused by adenovirus and group A and C rotaviruses.

Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.

A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.