(1) Rotavirus infection in adults: molecular and serologic characterization of the isolates
Osamu Nakagomi - Akita University School of Medicine
Previously we found that about 15% of adult diarrhea cases were attributable to rotavirus infection but in that study rotavirus was identified by RotaClone alone. This studay was undertaken to confirm what strains they really were. Of >100 specimens collected in a four-month period, 11 were rotavirus positive by both ELISA and RT-PCR. There were four G9, four G2 and only one G1. We also examined neutralization antibody responses against homotypic and heterotypic strains. Both homotypic and heterotypic responses occurred and there was virtually no difference between them. Acute phase antibody titers measured by ELISA were not different between those in whom rotavirus was identified and those in whom diarrhea was present but rotavirus was not identified. So apparently adults who were infected with rotavirus did not represent those who had low levels of antibody.
(2) Diarrhea-inducing activity in suckling mice of group A avian rotavirus NSP4s
Yoshio Mori - Gifu University
Pigeon rotavirus PO-13, but not turkey strains Ty-1 and Ty-3 and chicken strain Ch-1, induce diarrhea in mice. The amino acid homologies of NSP4s between PO-13 and turkey strains are 90-97%, whereas Ch-1 NSP4 was 78-79% divergent from the other avian rotavirus NSP4s. Thus, there are two distinct avian rotavirus NSP4 genotypes. When expressed in E. coli, NSP4s from any avian rotavirus caused diarrhea in mice. Enterotoxin domain exists in aa 109-135 of PO-13 NSP4. This domain is conserved in terms of structural properties despite extremely low homologies in the full lengths of NSP4s between avian and mammalian rotaviruses. Avian rotavirus NSP4s have enterotoxigenic activity in suckling mice, irrespective of the virulence of the strain from which the NSP4 was derived.
(3) Mosaic structures of rotavirus VP4 genes generated after successive passages at high m.o.i.: implications of inter-segmental rearrangements
Kazuyoshi Kojima - Sapporo Medical University
Rearrangement is one mechanism of generating diversity in the rotavirus genome. We analysed the progenies with rearrangement of a bovine rotavirus strain UK, which were generated by a series of long-term passages at high m.o.i. After approximately 50 consecutive passages in culture, a range of rearrangements involving NSP2, NSP5 and VP4 genes were identified by PAGE. A ladder-like pattern of multi bands in a position adjacent to the authentic gene 4 was of particular interest. Formation of such multi-bands was resulted from accumulations of rearrangements, including a single or multiple deletions within the VP4 gene. Surprisingly, however, a half of the clones were made up of a combination of VP4 and NSP2 origins. This is the first report describing the occurrence of inter-segmental rearrangements in the rotavirus genome.
(4) Intranasal or oral immunization of mice with VP6, a new rotavirus vaccine candidate, consistently protects mice against viral shedding after rotavirus challenge
Richard Ward - Children's Hospital Medical Center, Cincinnati
Intranasal administration of a chimeric VP6 protein from the EDIM strain to adult BALB/c mice along with an attenuated mutant LT stimulated 99% reductions in rotavirus shedding after subsequent EDIM challenge. Intranasal immunization stimulated 99% reductions in EDIM shedding in four additional strains of inbred mice belonging to three haplotypes. Protection stimulated against EDIM antigen shedding following intranasal immunization with VP6 from the human CJN strain was 86%. Protection against EDIM shedding was also maintained after intranasal immunization of three strains of outbred mice with either EDIM or CJN VP6. Protection stimulated by oral immunization of BALB/c mice with two doses of chimeric VP6 plus LT was not significantly different from that induced by intranasal immunization. These results support further evaluation of VP6 as a vaccine.
(5) Recent studies of rotavirus replication and immunity
Harry Greenberg - Stanford University School of Medicine
Recent work in the transport and release of RRV from polarized intestinal Caco-2 cells has shown that RRV is released almost exclusively from the apical surface at late time points after infection but before any evidence of cellular lysis. In order to understand the release of RRV during infection, infected Caco-2 cells were radiolabeled with [35S] and chased for various times. The cells were then adjusted to 40% Optiprep and run on an Optiprep gradient. RRV structural proteins were detected in the RAFT containing fraction post chase, and became more plentiful with longer periods of chase. To determine if the increase of viral proteins in the RAFT fractions reflected an association of infectious rotavirus particles with RAFTS, we measured infectivity and genomic dsRNA in each fraction. An increase of infectivity as well as the presence of dsRNA was found in the RAFT fractions. Treatment of cells with drugs that interfere with RAFTS formation also alters the migration of RRV particles through the gradient and reduced the release of infectious particles to the cell surface. The association of RRV particles with rafts in situ prior to detergent extraction was investigated by double indirect immunofluorescence staining and confocal microscopy using antibodies against rotavirus proteins and detecting GM1 as a lipid RAFT marker. The colocalization of mature virus particles and GM1 was confirmed using mAb 159, a conformational dependent mAb that recognizes VP7 exclusively when it is assembled in virus particles. Taken together these results indicate that rotavirus infectious particles associate with RAFTS during replication, suggesting that rotavirus uses these microdomains for transport to the cell surface.
We have also carried out new studies directed at investigating the neutralization activity of IgA antibody directed at VP6. We found that this antibody can neutralize virus if directed to the cytoplasm of infected cells, that it blocks an early stage in replication and that its binding to VP6 blocks transcription. In other studies we have investigated the requirements for transcytosis for functional activity of protective VP6 antibodies.
In additional studies we are characterizing the expression of homing and chemokine receptors on B cells that express RV specific antibody on their surface in humans following RV infection using flow cytometry. Preliminary results show that during acute infection in children, RV circulating B cells are predominantly large lymphocytes with the a4b7+/CCR6-/CCR9+/CCR10+ phenotype. However, during the convalescent phase and in recently exposed adults, a mixed population of small and large lymphocytes predominantly a4b7+/CCR6+/CCR9-/CCR10- is observed. These profiles of homing and chemokine receptor expression are correlated with two functional subsets of B cells: gut-targeted antibody secreting cells and circulating post-germinal center memory cells. Further studies are in process to confirm these results and to better characterize these cells using memory and activation markers.
(6) Detection of group B human rotavirus in Bangladesh: clinical and epidemiologic features of diarrhea and phylogenetic analysis of the Bangladeshi group B rotavirus strain
Dr. Ahmed, M.U.- Bangladesh Agricultural University
We recently detected group B rotaviruses (GBRs) in Bangladesh mostly in adults with severe diarrhea. Of 959 and 192 stool specimens collected from diarrheal cases of adults and children, respectively, 14 rotaviruses showed RNA patterns typical of GBR. Twelve GBRs were found in specimens from adults aged from 27 to 60, while two viruses were derived from children. Nucleotide sequences were determined for a GBR designated Bang373 to examine its genetic relatedness to Chinese ADRV, Indian CAL-1, and animal GBRs. Bang 373 VP7 was 98% identical with CAL-1 VP7 gene and 92% identical with ADRV VP7, while identity with animal GBRs was much lower. Similar to VP7 gene, other Bang373 genes encoding VP2, VP4, VP6, NSP1-NSP5 showed much higher identities with those of CAL-1 (98-99%) than those of ADRV (91-94%). Moreover, Bang373 and CAL-1 shared some common characteristics which were not found in ADRV in deduced amino acid sequences of VP2, VP4, NSP2, and NSP3. These results indicated that Bang373 and CAL-1 are virtually identical to each other and that Bang373 (CAL-1)-like GBRs might have been widely distributed in the area around the Bay of Bengal.
(7) Fecal shedding of Norwalk virus and Sapporo virus in infants in Japan
Masazumi Tatsumi - Sapporo Medical University
In January and February 2000, there were two outbreaks of acute gastroenteritis caused by Norwalk virus (NV) and Sapporo virus (SV) in an infant home in Sapporo. Of these 36 infants (5-26 months old) involved in both outbreaks, 20 (aged 6 to 24 months old) had a NV gastroenteritis and 15 (aged 7 to 26 months old) had a SV gastroenteritis. The RT-PCR followed by Southern hybridization method was used for NV detection and the nested PCR method was used for SV detection. In NV gastroenteritis, virus shedding lasted from 1 to 22 days (mean 10.6 days) and was correlated to the clinical severity and inversely correlated with age. Pre-existing antibody titers to NV were lower in the clinically severe group than in the milder group and in the younger age group than in the older age group. In SV gastroenteritis, virus shedding lasted from 1 to 22 days (mean 6.6 days) and was not correlated with the clinical severity or age. The primary case of the outbreak was an asymptomatically-infected infant. Higher pre-existing antibody titers to SV were correlated with resistance against illness and infection. These children shed NV and SV into stool even after the recovery from illness. The presence of asymptomatic virus shedding complicates the prevention against nosocomial infection of NV and SV gastroenteritis in the closed community.
(8) Fine epitope mapping of the monoclonal antibodies against Norwalk viruses with broad reactivity
Tomoko Yoda - Osaka Prefectural Institute of Public Health
Broadly-reactive monoclonal antibodies are needed in diagnostic assays. Two monoclonal antibodies against the capsid protein of genogroup II Norwalk virus were shown to bind to an approximately 40 amino acid-length fragment (designated fragment 2) of genogroup I Norwalk virus as well as the fragment 2 of genogroup II Norwalk virus. These two monoclonal antibodies showed different yet broad reactivities to Norwalk virus capsid proteins. Epitope mapping was done by using 24 different sequences of E. coli-expressed peptides corresponding to the fragment 2 or whole recombinant capsid proteins. Both monoclonal antibodies recognized epitopes present on an 11-amino-acid residues within the fragment 2. One monoclonal antibody (designated 1B4) bound to a conserved 5-amino-acid stretch (i.e., IDPWI). The other monoclonal antibody (designated 1F6) did not bind to the conserved 5-amino-acid stretch or 5 different sequences of the 11-amino-acid stretch of 24 types. Different reactive patterns of these two monoclonal antibodies suggested that both 1B4 and 1F6 were useful for detecting broad range of Norwalk viruses.
(9) Molecular epidemiology of adenovirus among children with diarrhea in Japan, Vietnam and Korea
Hiroshi Ushijima - Graduate School of Medicine, The University of Tokyo
To determine the prevalence of adenovirus infection in Japan, Vietnam and Korea, 3,577 fecal specimens were collected from children with acute gastroenteritis under 15 years of age. Detection was done by ELISA and serotypes were determined by the combination of PCR and RFLP. Overall,
153/3,577(4.3%) specimens contained adenovirus. In Japan, 66/95 (69.4%) were typed as Ad41, the most common serotype, followed by Ad2 (9.5%), Ad5 (7.4%), Ad3 (4.2%), Ad8 (3.2%). Ad40 was detected 2 of 95 (2.1%). In Vietnam, Ad41 was the most common serotype (25/38, 65.8%). The relative frequency of Ad40 was 26.3% (10/38), and this was the second common serotype of adenovirus associated with diarrhea. Ad2, 5 were 1/38 (2.6%), 1/38 (2.6%), respectively. In Korea, Ad41 was predominant (10/20, 50%), while Ad3 and Ad8 were found in 6/20 (30%) and 4/20 (20%), respectively. Ad40 was not detected. Of note, 6 specimens from Vietnam belonged to uncommon Ad41 strains. They appeared to have a 45-bp deletion in the fiber gene found only in Ad40 strains.
The enteric adenovirus was the most important and common subgenus of adenovirus in diarrheal children, but the prevalence of Ad41 and Ad40 was different in Japan, Vietnam and Korea.
(10) The 5'-end sequence of the genome of Aichi virus, a picornavirus, contains an element critical for viral RNA replication and encapsidation
Jun Sasaki - Fujita Health University
Aichi virus, is a member of the genus Kobuvirus of the family Picornaviridae. Computer-assisted prediction of the secondary structure of the 5' end of the genome suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. In this study, we investigated function of this stem-loop structure in virus replication using various site-directed mutants derived from an infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5' end of the positive strand, but not the 3' end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which the 7-nucleotide stretches of the middle part of the stem were exchanged with each other to maintain the base pairings of the stem, had efficient RNA replication and translation abilities, but formed no plaques. Sedimentation analysis of viral and subviral particles generated in the cells transfected with this double mutant RNA revealed that this mutant has a defect in RNA encapsidation. These results indicate that the stem-loop structure at the 5' end of the Aichi virus genome is a bifunctional element involved in viral RNA replication and encapsidation.