Hainian Yan

Changing distribution of group A rotavirus G-types and genetic analysis of G9 circulating in Japan

Summary.A total of 1,797 fecal specimens from infants and children with acute gastroenteritis in Japan from July 2000 to June 2003 were tested for group A rotavirus by ELISA, RT-PCR, RNA-PAGE and latex agglutination methods. Of these, 439 were found to be positive for group A rotavirus and this presented 24.4%. In 2000–2001, G1 was the most prevalent (45.5%) followed by G2 (32.5%), G3 (12.3%), G9 (5.9%) and G4 (2.6%). However, G2 was found predominant with 40% in the following year (2001–2002). Interestingly, G9 had a rapid increase of infection up to 17.8%. In 2002–2003, G3 dominated over other G-types with 34%. Another interesting feature of the study was the demonstration of great genetic diversity among G9 strains in Japan. Worth of note was the first prevalence pattern of rotavirus G-types with an increase of G2, G3 as well as G9 and a decrease of G1 during the 20 year-survey of rotavirus infection in Japan.

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.

A novel RT-multiplex PCR for enteroviruses, hepatitis A and E viruses and influenza A virus among infants and children with diarrhea in Vietnam

Summary.A novel reverse transcription-multiplex polymerase chain reaction (RT-multiplex PCR) assay that can detect enteroviruses, hepatitis A and E viruses and influenza A virus from various hosts (avian species, human, swine and horse) was developed. The identification of that group of viruses was performed with the mixture of four pairs of published specific primers (F1 and R1, P3 and P4, 2s and 2as, MMU42 and MMU43) for amplifying viral genomes and specifically generated four different amplicon sizes of 440, 267, 146 and 219 bp for enteroviruses, hepatitis A and E viruses and influenza A virus, respectively. A total of 276 fecal specimens (previously screened for rotavirus, adenovirus, norovirus, sapovirus and astrovirus-negative) from infants and children admitted into hospital with acute gastroenteritis in Ho Chi Minh city, Vietnam during October 2002 and September 2003 were collected and further tested for the presence of those viruses by RT-multiplex PCR. Enteroviruses were identified in 27 specimens and this represented 9.8%. No hepatitis A and E viruses and influenza A virus was found among these subjects. The sensitivity and specificity of RT-multiplex PCR were also assessed and demonstrated the strong validation against RT-monoplex PCR. Taken together, the findings clearly indicated that this novel RT-multiplex PCR is a simple and potential assay for rapid, sensitive, specific and cost-effective laboratory diagnosis to investigate molecular epidemiology of acute gastroenteritis caused by enteroviruses, hepatitis A and E viruses and influenza A virus. This report is the first, to our knowledge, detecting these kinds of viruses in diarrheal feces from infants and children in Vietnam.

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. J. Med. Virol. 82: 878–885, 2010.

Novel recombinant norovirus in China.

To the Editor: Norovirus (NoV), the distinct genus within the family Caliciviridae, is a major cause of sporadic cases and outbreaks of acute gastroenteritis in humans (1). NoV possesses a positive-sense, single-stranded RNA genome surrounded by an icosahedral capsid. The NoV genome contains 3 open reading frames (ORFs). ORF1 encodes nonstructural proteins, ORF 2 encodes capsid protein (VP1), and ORF3 encodes a small capsid protein (VP2). NoV is still uncultivable by standard culture with different cell lines. However, expression of either VP1 or both VP1 and VP2 with recombinant baculoviruses formed viruslike particles that are morphologically and antigenically similar to the native virion (2). A fecal specimen was collected from an infant hospitalized with acute gastroenteritis in Kunming, China, in November 2004 and was tested for diarrheal viruses in a cooperative laboratory in Japan. The viral genome was extracted by using a Qiagen kit (Qiagen, Hilden, Germany). Polymerase chain reaction with specific primers resulted in the identification of astrovirus, rotavirus, sapovirus, adenovirus, and NoV genogroup I (GI) and GII (3). NoV polymerase was also amplified to identify recombinant NoV with primers Yuri22F and Yuri22R (4). Products were sequenced directly, and sequence analysis was performed by using ClustalX and SimPlot. The fecal specimen was positive for NoV GII. The Figure shows that the 146/Kunming/04/China sequence clustered into the distinct GII genotype 7 (Leeds/90/UK cluster). 146/Kunming/04/China was classified into the Saitama U4 cluster (GI/6) when polymerase-based grouping was performed. Altogether, 146/Kunming/04/China was expected to be the recombinant NoV with GII/7 capsid and GII/6 polymerase. Figure Changes in norovirus (NoV) genotypes on the basis of phylogentic trees of nucleotide sequences of 146/Kunming/04/China. Trees were constructed from partial nucleotide sequences of capsid and polymerase regions of 146/Kunming/04/China. 146/Kunming/04/China ... To eliminate the possibility of co-infection with 2 different NoV genotypes, to localize the potential recombination site, and to clarify a possible recombination mechanism, the ORF1/ORF2 overlap and flanking polymerase and capsid regions of 146/Kunming/04/China was amplified with primers Yuri22F and GIISKR to produce a 1,158-bp amplicon (3,4). When the sequence of 146/Kunming/04/China was compared with that of Saitama U4 by using SimPlot, a recombination site was found at the ORF1/ORF2 overlap. Before this junction, 146/Kunming/04/China and Saitama U4 were homologous. After the ORF1/ORF2 overlap, however, the homology was notably different. SimPlot showed a sudden drop in the nucleotide identity for 146/Kunming/04/China. ClustalX showed that 146/Kunming/04/China shared a high identity (93%) in the polymerase region and a low identity (78%) in the capsid region with Saitama U4. In contrast, high identity (95%) in the capsid region was found between 146/Kunming/04/China and Leeds/90/UK. Since Leeds/90/UK polymerase was not available in GenBank, the polymerase homology between 146/Kunming/04/China and Leeds/90/UK was unknown. Polymerase of 146/Kunming/04/China was almost identical with that of Saitama U4, but the capsids of 146/Kunming/04/China and Leeds/90/UK were distinctly different from that of Saitama U4. This genetic pattern of 146/Kunming/04/China implied a novel, naturally occurring recombinant NoV with GII/7 capsid and GII/6 polymerase. RNA recombination is a mechanism for virus evolution (5). Literature documenting recombination in NoV is fairly rich, but none is from China (6). Therefore, 146/Kunming/04/China was not only the first but also the first recombinant NoV from China. This isolate shared the closest sequences of polymerase and capsid with Saitama U4 and Leeds/90/UK, respectively. Strain Saitama U4 was detected in 1997 in Japan (7), whereas strain Leeds/90/UK was detected in 1990 in the United Kingdom (8). Quite possibly, Saitama U4 and Leeds/90/UK were parental strains of 146/Kunming/04/China. However, the distant geographic relationship of these strains obscured evidence of where and when the recombination event occurred. This phenomenon also suggested that these parent strains or this progeny strain might be more prevalent than is often assumed. Recombination depends on various immunologic and intracellular constraints. Recombinant viruses are all alike in that they successfully pass through 5 stages: 1) successful co-infection of a single host, 2) successful co-infection of a single cell, 3) efficient replication of both parental strains, 4) template switching, and 5) purifying selection (9). In this study, 146/Kunming/04/China was recovered from a patient with diarrhea, fever, and vomiting. This observation indicated that this strain theoretically fulfilled all prerequisites for recombination. The NoV capsid is predicted to be well suited for genotype classification (10). In this study, 146/Kunming/04/China belonged to 2 distinct genotypes, 7 and 6, by capsid- and polymerase-based groupings, respectively. Moreover, the recent demonstration of recombination in an increasing number of NoVs suggests that it is a more widespread event than was previously realized. Consequently, the phylogenetic classification of NoV on the basis of on capsid sequence is questionable. We suggest that classification of NoV strains should rely on not only capsid sequence but also polymerase sequence. In conclusion, our results described the genetic characterization of novel, naturally occurring recombinant NoV and increased evidence for the worldwide distribution of recombinant NoV. This report is the first to describe acute gastroenteritis caused by recombinant NoV in China and warns of the threat it poses.