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. © 2010 Wiley‐Liss, Inc.     

Isolation and molecular characterization of Aichi viruses from fecal specimens collected in Japan, Bangladesh, Thailand, and Vietnam

Aichi virus is a new member of the family Picornaviridae, genus Kobuvirus, and is associated with human gastroenteritis. This study detected Aichi virus in 28 of 912 fecal specimens which were negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus and were collected in Japan, Bangladesh, Thailand, and Vietnam during 2002 to 2005.     

Antigenic and genetic conservation of H3 influenza virus in wild ducks

The hemagglutinins of H3 influenza viruses isolated from migratory ducks on the Pacific flyway in Japan during the period 1977 to 1985 were analyzed antigenically and genetically. Antigenic analysis using monoclonal antibodies to the hemagglutinins of A/Aichi/2/68 (H3N2) and A/duck/Hokkaido/8/80 (H3N8) viruses showed that antigenic drift occurred extensively in human strains, whereas the hemagglutinins of duck viruses were highly conserved. It was also found that the hemagglutinins of duck viruses were antigenically closely related to that of human 1968 H3 prototype strains. Nucleotide sequence analysis of seven duck H3 hemagglutinin genes showed a limited number of changes among the six duck isolates and between these duck isolates and Aichi/68. The deduced amino acid sequence revealed amino acid changes randomly distributed throughout the molecule and not confined to antigenic sites. These findings indicate that the duck virus hemagglutinin genes are conserved in nature and that viruses of different lineages cocirculate.     

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.     

First molecular detection of Aichi virus in sewage and shellfish samples in the Monastir region of Tunisia

The aims of our investigations were (1) to look for Aichi virus in environmental samples and (2) to compare the Aichi virus strains in both clinical and environmental samples in order to evaluate the role of environmental contamination as a possible vehicle for viral transmission. Aichi virus was detected in 15 (6%) sewage samples and in 4 (6.6%) shellfish samples. Aichi virus was identified for the first time in water samples. Phylogenetic analysis revealed several clusters that occurred sequentially in time, suggesting some parallelism in the evolution of environmental and human strains. Aichi virus present in sewage reflects the viruses circulating in the community.     

Genetic diversity of influenza B virus: the frequent reassortment and cocirculation of the genetically distinct reassortant viruses in a community

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439-447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses.     

Isolation and Characterization of Influenza C Viruses in the Philippines and Japan

From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined.     

Evolutionary Analysis of Influenza C Virus M Genes

The previous study of the 25 hemagglutinin-esterase (HE) glycoprotein genes of influenza C viruses identified four discrete lineages represented by C/Yamagata/26/81, C/Aichi/1/81, C/Aomori/74 and C/Mississippi/80, respectively. Here we compared the M gene sequence among the 24 viruses isolated between 1964 and 1991. A phylogenetic analysis showed that these genes have evolved into three distinct lineages. Lineage I included most of viruses with the HE genes of C/Yamagata/26/81-related lineage. The predominant members of lineage II were viruses having the HE genes of either C/Aichi/1/81- or C/Mississippi/80-related lineage. Lineage III contained only C/Aomori/74. Phylogenetic positions of several strains (C/Yamagata/64, C/Kanagawa/1/76, C/Miyagi/77 and C/Nara/1/85) were different between the M and HE gene trees, suggesting that they are reassortants. Furthermore, phylogenetic relationships between C/Mississippi/80-like and C/Aichi/1/81-like viruses were much closer for the M gene than the HE gene, raising the possibility that these two virus groups are genetically related by a reassortment event. Nucleotide changes in the M genes occurred at about 7% positions with a uniform distribution throughout the molecules. However, the predicted amino acid sequence of the matrix protein (M1) was conserved almost completely among the isolates analyzed. The amino acid sequence of the second protein (CM2) encoded by M gene was also highly conserved, but was more divergent than the M1 protein sequence, suggesting that the two M gene products are evolving differently in response to selective pressures or structural and functional constraints. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human bocavirus infection in children with acute gastroenteritis in Japan and Thailand

A total of 329 fecal specimens, which had been known to be negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus, and which were collected from infants and children with acute gastroenteritis in Japan and Thailand during 2005-2008 were screened for human bocavirus (HBoV). HBoV was detected by PCR with a primer pair that amplified the NP1 region of its genome and was genotyped by sequencing of the VP1/VP2 region. Of the 329 samples tested, 6 (1.8%) were positive for HBoV. Of these, five samples were collected from Japan and one sample was from Thailand, and the detection rates of HBoV in each country were 2% and 1.2%, respectively. For the detected HBoV, the capsid VP1/VP2 gene of all HBoV strains was successfully sequenced. Four Japanese HBoV strains studied were clustered into group 1, while the remaining Japanese strain and a unique Thai strain belonged to group 2. No severe acute gastroenteritis associated with HBoV was noted. This study provides better understanding on the epidemiology of HBoV infections in children with acute gastroenteritis in Japan and Thailand.     

Regulatory effects of matrix protein variations on influenza virus growth

Summary Influenza virus A/WSN/33 forms large plaques (> 3 mm diameter) on MDCK cells whereas A/Aichi/2/68 forms only small plaques (< 1 mm diameter). Fast growing reassortants (AWM), isolated by mixed infection of MDCK cells with these two virus strains in the presence of anti-WSN antibodies, all carried the M gene from WSN. On MDCK cells, these reassortants produced progeny viruses as rapidly as did WSN, and the virus yield was as high as Aichi. The fast-growing reassortants overcame the growth inhibitory effect of lignins. Pulse-labeling experiments at various times after virus infection showed that the reassortant AWM started to synthesize viral proteins earlier than Aichi. Taken together, we conclude that upon infecting MDCK cells, the reassortant viruses advance rapidly into the growth cycle, thereby leading to an elevated level of progeny viruses in the early period of infection. Possible mechanisms of the M gene involvement in the determination of virus growth rate are discussed, in connection with multiple functions of the M proteins.     

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.     

Origin of the hemagglutinin gene of H3N2 influenza viruses from pigs in China

Influenza viruses of the H3N2 subtype similar to Aichi/2/68 and Victoria/3/75 persist in pigs many years after their antigenic counterparts have disappeared from humans (Shortridge et al. (1977). Science 19, 1454-1455). To provide information on the mechanism of conservation of these influenza viruses in pigs, the hemagglutinin (HA) of four isolates from swine derived from Taiwan and Southern China were analyzed antigenically and genetically. The reactivity pattern of these viruses with a panel of monoclonal antibodies indicates that the HAs of these swine viruses were antigenically closely related to duck H3 and early human H3 viruses. Sequence analysis of the H3 genes from three swine viruses revealed that the swine H3 genes are more closely related to the duck genes than to early human H3 virus (A/Aichi/2/68). The degree of sequence homology of these genes is extremely high (more than 96.5%). Furthermore, the deduced amino acid sequence of the three swine HAs at residues 226 to 228 in the proposed receptor-binding site is Gln-Ser-Gly and is common with the majority of avian influenza viruses. These findings indicate that these H3 viruses may have been introduced into pigs from ducks. The HA gene of the fourth swine influenza virus from Southern China was genetically equally related to avian and early human H3 strains although the sequence through the receptor-binding pocket (226-228) was typical of a human H3 virus, suggesting that either this swine HA gene was derived from ducks or an early human H3 virus was introduced into the pig population where the virus accumulated substantial mutations. The present strains revealed genetic heterogeneity of swine H3 influenza viruses in nature.     

Extinction and rapid emergence of strains of dengue 3 virus during an interepidemic period

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.     

在兰州地区儿童粪便中检测Aichi virus

目的 从兰州腹泻和正常儿童粪便标本中检测 Aichi virus,同时探讨 Aichi virus 与婴幼儿腹泻之间的联系.方法 根据文献发表资料,采用RT-PCR方法扩增 Aichi virus 3CD 片段,阳性产物经测序确定,并与已发表的该病毒序列进行比对分析.结果 在46份腹泻住院患儿粪便标本和299份腹泻门诊就诊患儿标本中各检出1例 Aichi virus,总检出率为0.06%,正常对照儿童中未检测到 Aichi virus.2株病毒3CD区基因与已知参考株核苷酸序列同源性均为97%,系统进化分析显示这2株病毒属于B基因型.结论 我国存在B基因型的 Aichi virus,但要明确我国 Aichi virus 的病原学及流行病学特点需要更多研究.     

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

Of five globally important VP7 (G) serotypes (G1-4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.     

Seroprevalence of Aichi Virus in a Spanish Population from 2007 to 2008

Viruses are among the most common causes of acute gastroenteritis. In recent years, new viruses causing outbreaks of acute gastroenteritis have been described. Among these, Aichi virus was identified in Japan in 1989. Aichi virus belongs to the Kobuvirus genus in the family Picornaviridae. This virus has been detected in outbreaks of gastroenteritis associated with oyster consumption and in pediatric stool samples, but little is known about its epidemiology or pathogenesis. In the present study, the prevalence of antibodies to Aichi virus in a Spanish population was determined between 2007 and 2008 by using an enzyme-linked immunosorbent assay (ELISA). As in previous studies, a high seroprevalence of antibodies to Aichi virus (70%) was observed, with levels differing according to age. We observed significant differences in titers of antibody to Aichi virus among different age groups, grouped by decades. We report high ELISA and neutralizing antibody titers, and both titers fitted a sigmoid curve significantly. However, this virus is seldom detected; therefore, further studies are needed to gain a better understanding of its importance as a pathogenic agent.Viruses are a common cause of gastroenteritis and affect humans of all ages. Rotavirus (mainly group A), calicivirus (including norovirus and sapovirus), adenovirus, and astrovirus are considered the major causes of viral gastroenteritis. However, in many cases of gastroenteritis, no specific pathogen can be identified, and other viruses, such as Aichi virus, may be involved. This virus was proposed as a probable cause of nonbacterial gastroenteritis associated with oyster consumption in Aichi, Japan, in 1989 (16). Aichi virus belongs to the genus Kobuvirus, in the family Picornaviridae (9, 19). The major differences between the genus Kobuvirus and other genera of the same family are found in the coding region of the L protein, in the absence of a VP0 cleavage site, and in the distinct morphology of the 2A protein (14).The Aichi virus genome is a single-stranded, positive-sense RNA molecule of 8,260 nucleotides and has a poly(A) tail. The single large open reading frame encodes a polyprotein of 2,432 amino acids. This polyprotein, like those of the other members of the family, is cleaved into the structural proteins VP0, VP3, and VP1 and the nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D (11, 19). Aichi virus has been classified into two genotypes (A and B) by phylogenetic analysis of a 519-bp sequence at the 3C-3D (3CD) junction (20), and recently (in 2008), a new genotype, C, was proposed by Ambert-Balay et al. (1). These authors have also genotyped Aichi virus based on phylogenetic analysis of a 699-bp sequence of the gene encoding the VP1 protein. The results of this analysis correlate well with the 3CD sequence classification and also give rise to A, B, and C genotypes.Little is known about the incidence of Aichi virus infection in humans. Aichi virus antigen or viral RNA was first detected in fecal samples collected in Japan (17). The virus was later isolated from patients with gastroenteritis, comprising Pakistani children and Japanese travelers from Southeast Asia (18), and among patients from Japan, Bangladesh, Thailand, and Vietnam (8). In 2006, the virus was isolated for the first time in the Americas (Brazil) and Europe (Germany) (7), and since then, Aichi virus has been detected in France (6), Tunisia (12, 13), Hungary (10), and Finland (5).The first study of Aichi virus seroprevalence was performed in Japan and revealed a high rate of antibodies to Aichi virus (17). Other studies in Germany (7) and in France (3) have given similar results.The purpose of the present study was to determine the seroprevalence of antibodies to Aichi virus in Valencia, Spain, during the years 2007 to 2008.     

Prevalence of newly isolated, cytopathic small round virus (Aichi strain) in Japan.

Cytopathic small round virus (Aichi strain), isolated from a patient with oyster-associated gastroenteritis, showed no reaction in the polymerase chain reaction method for enteroviruses or in the enzyme-linked immunosorbent assay (ELISA) for the five serotypes of astroviruses. Our ELISA was sensitive in detecting the Aichi strain antigen in stool samples, but there was no reaction in this ELISA with any non-Aichi strains of enteric viruses, with such origins as enterovirus, rotavirus, Norwalk virus, calicivirus, or astrovirus. In the ELISA, 13 of 47 stool samples from adult patients in five of nine oyster-associated gastroenteritis outbreaks were positive, but only 1 of 397 pediatric stool samples in Aichi Prefecture was positive. The prevalence rate for Aichi strain antibody was found to be 7.2% for persons aged 7 months to 4 years. The prevalence rate for antibody to Aichi strain increased with age, to about 80% in persons 35 years old. On the basis of the results of the present study, it was hypothesized that Aichi strain could be a new type of small round virus that mainly produces diarrhea in patients in the 15- to 34-year-old age group, 50 to 76% of whom possess neutralizing antibody.