Aichi Virus Research Articles

Aichi Virus 2A Protein Is Involved in Viral RNA Replication

The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative- and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis.

Bovine Kobuviruses from Cattle with Diarrhea

To the Editor: A new species of kobuvirus, named U-1 strain, was first recognized in 2003 as a cytopathic contaminant in a culture medium of HeLa cells that had been used for >30 years in the laboratory (1). The RNA genome of the U-1 strain comprises 8,374 nucleotides; the genome organization is analogous to that of picornaviruses. Morphologically, the U-1 strain resembles the Aichi virus, but genetically it is distinct (1). Therefore, the U-1 strain is classified as a new species of genus Kobuvirus in the family Picornaviridae, and it is called bovine kobuvirus (1). To date, the genus Kobuvirus consists of 2 species, Aichi virus and bovine kobuvirus (2). The Aichi virus is associated with acute gastroenteritis in humans (3–5); bovine kobuvirus infection has been detected only in cattle (1). Only 1 report has described the discovery and epidemiologic features of bovine kobuvirus (1). Of serum samples from 72 healthy cattle, 43 (59.7%) were positive for neutralizing antibody against bovine kobuvirus U-1 standard strain at a titer of >16. In addition, 12 (16.7%) of 72 stool samples collected from the cattle were positive for the bovine kobuvirus genome by reverse transcription–PCR (RT-PCR) (1). This finding suggested that bovine kobuvirus is common and that the virus particles could be detected in the stool samples of infected cattle. We therefore conducted an epidemiologic survey of bovine kobuvirus and report detection of this virus in stool samples from calves with diarrhea during 2001–2004 in Chiang Mai Province, Thailand. From November 2001 to July 2004, a total of 72 fecal specimens were collected. The age of the calves ranged from 7 to 49 days. The presence of bovine kobuvirus in fecal specimens was detected by using RT-PCR with a protocol modified from the method described by Yamashita et al. (1). All the bovine kobuvirus strains detected in our study were analyzed further by direct sequencing of their PCR amplicons with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an automated sequencer (ABI 3100, Applied Biosystems). The nucleotide sequences of these portions were compared with those of reference strains available in the GenBank database by using BLAST (6). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version 3.1 (7). The nucleotide sequences of bovine kobuvirus strains described in this study were deposited in GenBank under accession nos. {type:entrez-nucleotide-range,attrs:{text:EF659450-EF659455,start_term:EF659450,end_term:EF659455,start_term_id:151299687,end_term_id:151299697}}EF659450-EF659455. The bovine kobuvirus was detected by the RT-PCR screening method in 6 (8.3%) of the 72 fecal specimens collected. The partial 3D regions of all 6 bovine kobuviruses exhibited highly conserved sequences of 99.3%–100% nucleotide and 100% amino acid identities to each other. In addition, by searching for the closest sequences in the databank, we found that these sequences were closely related to all 13 bovine kobuvirus reference strains available in the GenBank database (U-1, K-35, K-36, K-49, K-38, N-5, K-3, K-4, K-6, K-60, N-2, K-44, and K-55); nucleotide and amino acid sequence identities ranged from 91.0% to 95.0% and 97.4% to 99.4%, respectively. Phylogenetic analysis of partial 3D nucleotide sequences of our bovine kobuvirus strains, together with those of all published bovine kobuvirus reference strains available in the GenBank database, is shown in the Figure. The Aichi virus standard strain was included in the tree as an outlier virus. The phylogenetic tree confirmed that all 6 virus strains belonged to bovine kobuvirus and formed a tight cluster in a monophyletic branch with other published bovine kobuvirus reference strains, but they were distantly related to the Aichi virus standard strain. Figure Phylogenetic analysis of the partial nucleotide sequence encoding the 3D region of bovine kobuviruses isolated in this study and other reference strains recognized to date. The tree was generated on the basis of the neighbor-joining method using the MEGA ... Detection and characterization of bovine kobuvirus strains from different geographic areas are important for understanding the worldwide distribution, heterogeneity, and association of bovine kobuvirus with enteric disease in cattle. Our findings indicate the role of kobuviruses in diarrheal disease in cattle and provide additional information on their relationship to bovine kobuviruses reported previously.

Sequence analysis of the capsid gene of Aichi viruses detected from Japan, Bangladesh, Thailand, and Vietnam

Sequence analysis of the capsid gene of Aichi viruses was performed on 12 strains detected in Japan, Bangladesh, Thailand, and Vietnam during 2002-2005. The phylogenetic tree constructed from 17 nucleotide sequences of the capsid gene of the strains studied and reference strains demonstrated that Aichi virus strains clustered into two branches. A classification of Aichi viruses based on the capsid gene was proposed, in which lineage I consists of the Aichi virus strains detected from Japan, Thailand, Vietnam, and Germany, and lineage II includes Bangladeshi strains and a Brazilian strain.

UV Light Inactivation of Hepatitis A Virus, Aichi Virus, and Feline Calicivirus on Strawberries, Green Onions, and Lettuce

A majority of illnesses caused by foodborne viruses are associated with fresh produce. Fruits and vegetables may be considered high-risk foods, as they are often consumed raw without a specific inactivation step. Therefore, there is a need to evaluate nonthermal treatments for the inactivation of foodborne pathogens. This study investigates the UV inactivation of three viruses: feline calicivirus (a surrogate for norovirus), and two picornaviruses, hepatitis A virus and Aichi virus. Three produce types were selected for their different surface topographies and association with outbreaks. Green onions, lettuce, and strawberries were individually spot inoculated with 107 to 109 50% tissue culture infective doses (TCID50) of each virus per ml and exposed to UV light at various doses (≤240 mW s/cm2), and viruses were eluted using an optimized recovery strategy. Virus infection was quantified by TCID50 in mammalian cell culture and compared with untreated recovered virus. UV light applied to contaminated lettuce resulted in inactivation of 4.5 to 4.6 log TCID50/ml; for contaminated green onions, inactivation ranged from 2.5 to 5.6 log TCID50/ml; and for contaminated strawberries, inactivation ranged from 1.9 to 2.6 log TCID50/ml for the three viruses tested. UV light inactivation on the surface of lettuce is more effective than inactivation on the other two produce items. Consistently, the lowest results were observed in the inactivation of viruses on strawberries. No significant differences (P > 0.05) for virus inactivation were observed among the three doses applied (40, 120, and 240 mW s/cm2)on the produce, with the exception of hepatitis A virus and Aichi virus inactivation on green onions, where inactivation continued at 120 mW s/cm2 (P < 0.05).

Seroprevalence distribution of Aichi virus among a French population in 2006–2007

Little is known about the epidemiology of Aichi virus, which is a new member of the family Picornaviridae, in the genus Kobuvirus. We report here on seroprevalence in France. Sera were screened using an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G. Of 972 sera tested, seroprevalence ranged from 25% for the 7-month-to-9-year-old age group to about 85% for the 30-to-39-year-old age group and older age groups. Our ELISA correlated well with the microneutralization technique. This study shows that Aichi virus is quite frequent in France and that seroconversion occurs before the age of 40.

Interaction between Polypeptide 3ABC and the 5′-Terminal Structural Elements of the Genome of Aichi Virus: Implication for Negative-Strand RNA Synthesis

Secondary structural elements at the 5' end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5' end of the genome is required for negative-strand synthesis. We have previously shown that the 5'-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5'-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5'-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5'-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5'-terminal region did not correlate with the RNA replication ability.

Acute Infantile Gastroenteritis Associated with Human Enteric Viruses in Tunisia

This prospective study, conducted from January 2003 to June 2005, investigated the incidence and the clinical role of various enteric viruses responsible for infantile gastroenteritis in 632 Tunisian children presenting in dispensaries (380 children) or hospitalized (252 children) for acute diarrhea. At least one enteric virus was found in each of 276 samples (43.7%). A single pathogen was observed in 234 samples, and mixed infections were found in 42 samples. In terms of frequency, rotavirus and norovirus were detected in 22.5 and 17.4% of the samples, respectively, followed by astrovirus (4.1%), Aichi virus (3.5%), adenovirus types 40 and 41 (2.7%), and sapovirus (1.0%). The seasonal distribution of viral gastroenteritis showed a winter peak but also an unusual peak from May to September. The severity of the diarrhea was evaluated for hospitalized infants. No significant differences were observed between rotavirus and norovirus infections with regard to the incidence and the clinical severity of the disease, especially in dehydration.

Prevalence and Genetic Diversity of Aichi Virus Strains in Stool Samples from Community and Hospitalized Patients

Aichi virus has been proposed as a causative agent of gastroenteritis. A total of 457 stool specimens from children hospitalized with acute diarrhea and 566 stool specimens from adults and children involved in 110 gastroenteritis outbreaks were screened for the presence of Aichi virus by reverse transcription-PCR (RT-PCR) amplification of the genomic region of the 3C and 3D (3CD) nonstructural proteins. Our results show a low incidence of Aichi virus in pediatric samples and the existence of mixed infections with other microbiological agents in some cases. From the outbreak survey, it appears that the presence of Aichi virus is an indicator of mixed infections causing gastroenteritis outbreaks and that it could be involved in half of the oyster-associated outbreaks. A second RT-PCR was developed to amplify a part of the VP1 gene. The phylogenetic analysis showed a good correlation between the two classifications based on 3CD and VP1 gene sequences and revealed the prevalence of genotype A in France. It also allowed us to partially describe an Aichi virus strain that could represent a new genotype, thus suggesting the existence of a certain diversity.

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.

Etiological Role of Viruses in Outbreaks of Acute Gastroenteritis in The Netherlands from 1994 through 2005

Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.

Analysis of Aichi virus replication

Aichi virus is a member of the Family Picornaviridae. This virus was first isolated in 1989 from a stool specimen from a patient with oyster-associated gastroenteritis in Aichi, Japan. We analyzed the function of the 5' terminal region of the genome and the leader protein in virus replication. The results indicate that both the 5' terminal region of the genome and the leader protein are involved in viral RNA replication and encapsidation.

An RNA extraction protocol for shellfish-borne viruses

The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV-1), and the norovirus surrogate, feline calicivirus (FCV) strain KCD, for the purpose of RT-PCR detection within seeded oyster ( Crassostrea virginica) extracts. The RT-PCR equivalent sensitivities observed within seeded oysters as compared to virus stocks were 0.68, 6.8, 26, 5.6, and 14.5 RT-PCR 50 units when assaying 10% of total RNA extracted from seeded oyster extracts for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. For oysters exposed to virus-contaminated seawater, the detection equivalent sensitivities observed were 680, 68, 2600, 560, and 14.5 RT-PCR 50 for CAV9, CBV5, AiV and FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornaviruses and caliciviruses that could potentially contaminate shellfish.

Molecular characterization of the first Aichi viruses isolated in Europe and in South America

The occurrence of Aichi virus, a picornavirus associated with acute gastroenteritis, has so far only been described in Asian countries. This study reports the first finding of Aichi virus in clinical specimens from Germany and Brazil. The nucleotide sequences of both a German and a Brazilian isolate were determined, analyzed, and compared to known Aichi sequences. The German strain turned out to be a member of genogroup A, while the Brazilian belonged to genogroup B. For a primary assessment of the epidemiological importance of Aichi virus in Germany, a panel of 485 German serum samples was screened for antibody to Aichi virus, and a seroprevalence of 76% was found.

Macrophage-mediated inhibitory effect of Zingiber officinale Rosc, a traditional oriental herbal medicine, on the growth of influenza A/Aichi/2/68 virus.

The inhibitory effect of Zingiber officinale Rosc (ZOR), an Oriental traditional herbal medicine, on the growth of influenza A/Aichi/2/68 (Aichi) virus was investigated in Madin-Darby canine kidney (MDCK) cells. Direct addition of ZOR (0.1 approximately 100 microg/ml) to the infected cells did not have any inhibitory effect. However, the ZOR-induced conditioned medium (ZOR-CM) of RAW cells, a murine macrophage (Mphi) cell line, exhibited an apparent inhibitory effect on MDCK cells without cytotoxicity. In accordance with the time-dependent inhibitory effect of ZOR-CM, it has been demonstrated that tumor necrosis factor (TNF)-alpha was gradually accumulated in ZOR-CM by the induction of TNF-alpha mRNA expression in ZOR-stimulated RAW cells. Conversely, the inhibitory effect of ZOR-CM was reduced significantly by the removal of TNF-alpha after the formation of an immune complex with anti-TNF-alpha monoclonal antibody. These data suggested that ZOR itself has no inhibitory effect on the growth of influenza virus, but could exert its effect via macrophage activation leading to production of TNF-alpha.

The 5′-Terminal Region of the Aichi Virus Genome Encodes cis -Acting Replication Elements Required for Positive- and Negative-Strand RNA Synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.

Detection of Human Picornaviruses by Multiplex Reverse Transcription-PCR and Liquid Hybridization

A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5' untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.

Inactivation of selected picornaviruses by high hydrostatic pressure

The potential of high hydrostatic pressure processing (HPP) to inactivate Aichi virus (AiV), human parechovirus-1 (HPeV-1), and the coxsackievirus strains A9 and B5 was investigated. For coxsackievirus A9 (CAV9), a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented with 10% fetal bovine sera (FBS) resulted in 3.4-, 6.5-, and 7.6-log10 tissue culture infectious dose (50%) (TCID50) reductions at 400, 500, and 600megaPascals (MPa), respectively. For HPeV-1, a 5-min treatment in MEM with 10% FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log10 TCID50 at 400, 500, and 600MPa, respectively; however, AiV and coxsackievirus B5 (CBV5) in MEM supplemented with 2 and 10% FBS, respectively, remained fully infectious after a 5-min treatment at 600MPa. These data establish that different picornaviruses have widely variable pressure inactivation thresholds in response to HPP.

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

Aichi Virus Leader Protein Is Involved in Viral RNA Replication and Encapsidation

Isolation and characterization of a new species of kobuvirus associated with cattle

A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaviruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi virus. The virus sequence had 33 and 75 % amino acid identity with the Aichi virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other PICORNAVIRIDAE: The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus Kobuvirus but is distinct from Aichi virus. Of 72 cattle sera, 43 (59.7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16.7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus Kobuvirus, now termed bovine kobuvirus.

Aichi virus leader protein is involved in viral RNA replication and encapsidation.

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.