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Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density. Methods To measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate. Results In AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2. Conclusion We established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

Limited data are available on breakthrough fungemia, defined as fungemia that develops on administration of antifungal agents, in patients with hematological disorders. We reviewed the medical and microbiological records of adult patients with hematological diseases who had breakthrough fungemia between January 2008 and July 2019 at Toranomon Hospital and Toranomon Hospital Kajigaya in Japan.

Two novel actinobacteria, designated NBRC 107696T and NBRC 107697T, were isolated from sludge samples from a wastewater treatment plant and their taxonomic positions were investigated by a polyphasic approach. The cells of the strains were aerobic, rod-shaped, non-motile and non-endospore-forming. The strains contained glutamic acid, alanine and meso-diaminopimelic acid in the peptidoglycan. Galactose and arabinose were detected as cell-wall sugars. The predominant menaquinone was identified as MK-9(H2) and the major fatty acids were C16 : 0, C18 : 1ω9c and C16 : 1ω7c. The DNA G+C contents of NBRC 107696T and NBRC 107697T were 68.07 and 68.99 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that NBRC 107696T and NBRC 107697T were a clade with members of the genus Gordonia . The highest 16S rRNA gene sequence similarity values were obtained with Gordonia araii IFM 10211T (98.9 %) for NBRC 107697T, and Gordonia malaquae IMMIB WWCC-22T, Gordonia neofelifaecis AD-6T and Gordonia humi CC-12301T (98.1 %) for NBRC 107696T, respectively. The digital DNA–DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel separate species. The names proposed to accommodate these two strains are Gordonia spumicola sp. nov. and Gordonia crocea sp. nov., and the type strains are NBRC 107696T (=IFM 10067T=TBRC 11239T) and NBRC 107697T (=IFM 10881T=TBRC 11240T), respectively.

Aspergillus fumigatus is a critical human fungal pathogen that infects the host via inhalation of airborne conidia. These conidia then germinate to form filamentous hyphae, which secrete various elements to survive in the host lung.Elements such as proteins secreted by A. fumigatus can act as virulence factors in host tissues. Among secreted proteins, we were interested in the thaumatin-like proteins of A. fumigatus. In our analysis of the function of thaumatin-like proteins, we found that, like CalA and CalB, CalC has a secreted form. Originally, CalC was predicted to be a GPI-anchored protein, as documented in the Aspergillus Genome Database. Here, we report on a novel secreted form of CalC. Furthermore, we established two novel hybridomas, C103 and C306, which recognized CalC. Monoclonal antibodies produced by these hybridomas responded to recombinant CalC produced by the mammalian cell line HEK293T and to the supernatant of cultured A. fumigatus.Taken together, our data suggest that calC can be spliced to give rise to a novel secretory form of CalC, which is present in the supernatant of cultured A. fumigatus. The hybridomas that we established will be helpful in understanding the biological role of A. fumigatus CalC.

In recent years, Deep Learning, a Machine Learning algorithm, has been successfully used in many fields, and there have been reports of such a development in the field of behavior recognition. However, in general, high performance hardware is required to execute Deep Learning models in real-time. In this paper, we propose a surveillance camera system for behavior recognition using a self-organizing map (SOM) based on a time-series inference skeleton extracted from image data acquired by a monocular camera using a Machine Learning algorithm on an edge AI processor. The proposed surveillance camera system can be considered as a hybrid twin system. We performed skeleton extraction on an edge AI processor and evaluated experimentally in terms of processing performance and power consumption, and experimented with and evaluated an action recognition method using time-series inference skeleton information. An experimental evaluation of skeleton extraction on an edge AI processor shows the usefulness of the edge AI processor for use in a surveillance camera system. Experimental evaluation of action recognition by SOM showed that it is possible to recognize basic actions. We also found that each region on the U-matrix needs to be clarified in order to improve the percentage of correct answers.

The hammering sound inspection for pieces plays the Digital Twin part in the Hybrid Twin approach. The map generated by Self-Organizing Map (SOM) algorithm makes it possible to cluster the pieces into defected and non-defected groups by checking the corresponding hammering sounds. However, the applicability of map generated by SOM has not yet been discussed. This paper suggests a procedure how to improve a map generated by SOM algorithm. It shows that if the hammering sound of a piece is mapped on the boundary between the areas corresponding to defected and non-defected products, the map cannot be applied to inspect this piece and another map must be generated by repeating the training including the hammering sounds of such pieces by SOM algorithm. By executing the procedure some number of times, it is likely that the improved map will be used for hammering sound inspection.

Cutaneous cryptococcosis is classified as localized cutaneous cryptococcosis and cutaneous manifestations of disseminated cryptococcosis. The former presents as lesions, confined to isolated parts of the skin, which are neither systemically disseminated nor associated with cryptococcal fungemia or antigenemia. The latter presents as lesions through dissemination of Cryptococcus from visceral organs such as the lungs, with most cases being immunosuppressed hosts. We report the case of an immunocompetent elderly long-term pigeon fancier who presented with disseminated cutaneous cryptococcosis caused by Cryptococcus neoformans. Although the patient had been at risk of inhaling the pathogen by keeping pigeons for many years, and had been treated with topical steroids for a localized nodular lesion, the cause of development of multiple skin lesions could not be determined. The patient paradoxically showed no pulmonary or central nervous system symptoms, fungemia or glucuronoxylomannan antigenemia. Treatment with oral itraconazole 200 mg/day was not effective, but combination therapy of 5-fluorocytosine 200 mg/kg per day and fluconazole 100 mg/day resolved the disease.

Aspergillus fumigatus is a critical human fungal pathogen that infects the host via inhalation of airborne conidia. These conidia then germinate to form filamentous hyphae, which secrete various elements to survive in the host lung.Elements such as proteins secreted by A. fumigatus can act as virulence factors in host tissues. Among secreted proteins, we were interested in the thaumatin-like proteins of A. fumigatus. In our analysis of the function of thaumatin-like proteins, we found that, like CalA and CalB, CalC has a secreted form. Originally, CalC was predicted to be a GPI-anchored protein, as documented in the Aspergillus Genome Database. Here, we report on a novel secreted form of CalC. Furthermore, we established two novel hybridomas, C103 and C306, which recognized CalC. Monoclonal antibodies produced by these hybridomas responded to recombinant CalC produced by the mammalian cell line HEK293T and to the supernatant of cultured A. fumigatus.Taken together, our data suggest that calC can be spliced to give rise to a novel secretory form of CalC, which is present in the supernatant of cultured A. fumigatus. The hybridomas that we established will be helpful in understanding the biological role of A. fumigatus CalC.

Streptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified.Effects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography.Assam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of10kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances10kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation.The higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.

Next-generation technologies have prompted efforts towards generating a large repertoire of whole-genome sequences. The dermatophyte Arthroderma vanbreuseghemii has been considered as a good model in which to conduct molecular biological studies on this fungal group. Despite the considerable repertoire of molecular tools developed for this fungus, the lack of genomic data has represented a major limitation, preventing effective implementation of those tools. Herein, the authors report the first draft whole-genome sequence of this dermatophytic species. The size of the draft genome was 23 Mb, exhibiting a GC content of 48.1%. Given the significance of secreted proteases in tissue invasion, a comparative analysis of genes encoding extracellular proteases was performed between A. vanbreuseghemii and other dermatophytes. Furthermore, genes that might be involved in DNA repair also were compared among dermatophytes. Moreover, the complete mitochondrial genome of A. vanbreuseghemii was obtained and shown to consist of 24,287 bp with a GC content of 24%. In conclusion, the availability of genomic data for A. vanbreuseghemii is expected to facilitate the implementation of the molecular tools established for this fungus, enhancing our understanding of the biology of dermatophytes.

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains.

Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked to low susceptibility in azole-resistant isolates have previously been reported, few studies have been conducted to conclusively demonstrate the contribution of SNPs to decreased azole susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations Gly138Ser (GGC→AGC) and Asn248Lys (AAT→AAA) compared with the cyp51A of azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole susceptibility was slightly decreased when a Ser138 mutation was introduced into the azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole resistance.

The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f−/− mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f−/− mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds. Here, we describe the features of A. missouriensis 431(T), together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.

A new dimeric sesquiterpenoid with two eudesmane skeletons through a urea linkage, halichonadin A ( 1 ), as well as three new eudesmane sesquiterpenoids having a carbamate, an isonitrile, or an amino functionality, halichonadins B ( 2 ), C ( 3 ), and D ( 4 ), respectively, have been isolated from a marine sponge Halichondria sp., and the gross structures and relative stereochemistry of 1 – 4 were elucidated on the basis of spectral data and chemical means.

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1 . Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4 . Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1 .

We demonstrated that the rox gene of Nocardia farcinica encodes a rifampicin monooxygenase capable of converting rifampicin to a new compound 2'-N-hydroxy-4-oxo-rifampicin with a markedly lowered antibiotic activity. The deletion mutation (Deltarox) of the rox gene gave no significant influence to the rifampicin resistance of N. farcinica. However, transformation with a plasmid containing an overexpressing the rox gene markedly raised the rifampicin resistance in the strain with the deletion mutation of the rpoB2 gene as the principal rifampicin resistance determinant. On the other hand, rifampicin was decolorized by the wild-type strain, whereas it remained intact when incubated with the Deltarox strain. Based on these results, it will be conclusive that the rox gene is capable of initiating rifampicin degradation with a new metabolite formation at the first step and having a role as the secondary rifampicin resistance factor in N. farcinica.

Background. In a study performed in 1983, 18 adult volunteers received oral challenge with the virulent human rotavirus strain D (G1P1A[8],NSP4[B]). To identify correlates of resistance to rotavirus infection, we analyzed levels of serum immunoglobulin (Ig) A and IgG antibodies to various rotaviral antigens in 16 of the 18 volunteers. Methods. We used immunocytochemical assays that involved a total of 16 different recombinant baculoviruses, with each baculovirus expressing one of the following major serotype/genotype rotavirus proteins for the serologic assays: (1) viral protein (VP) 4 with P1A[8], P1B[4], P2A[6], P3[9], or P4[10] specificity; (2) VP7 with G1-G4 or G9 specificity; and (3) nonstructural viral protein (NSP) 4 with genotype A, B, C, or D specificity. Results. The prechallenge titers of IgG antibody to VP7 types G1, G3, G4, and G9; VP4 types P1A[8], P1B[4], P2A[6], and P4[10]; and NSP4 type [A] in the group of noninfected volunteers (n = 11) were significantly higher than those in the group of infected volunteers (n = 5; of these 5 volunteers, 4 were symptomatically infected). Moreover, logistic regression analysis showed that resistance to rotavirus infection most closely correlated with higher prechallenge titers of IgG antibody to homotypic VP7 (G1) and VP4 (P1A[8]). Conclusions. These results suggest that protection against rotavirus infection and disease is primarily VP7/ VP4 homotypic and, to a lesser degree, heterotypic.

Nocardithiocin is a novel thiopeptide compound produced by the pathogenic Nocardia pseudobrasiliensis strain IFM 0757. It shows a strong activity against acid-fast bacilli such as the Mycobacterium and Gordonia species. Nocardithiocin was highly active against rifampicin-resistant as well as -sensitive Mycobacterium tuberculosis strains, and most of the resistant strains were inhibited at concentrations ranging from 0.025 to 1.56 microg ml(-1). The structure of the thiopeptide antibiotic containing thiazole, natural and unnatural amino acids was elucidated by MS and NMR spectral analyses.

Using an immunocytochemical staining assay involving six different recombinant baculoviruses with each expressing one of the major bovine rotavirus VP7 (G6, G8 and G10) and VP4 (P6[1], P7[5] and P8[11]) serotypes, we analyzed IgG antibody responses to individual proteins in archival serum samples collected from 31 calves monthly from 1 to 12 months of age during 1974-1975 in Higley, Arizona. Seroresponses to VP7 and VP4, as determined by a fourfold or greater antibody response, were not always elicited concurrently following infection: in some calves, (1) seroresponses to VP7 were detected earlier than to VP4 or vice versa; and (2) a subsequent second seroresponse was detected for VP7 or VP4 only. In addition, a second infection was more likely to be caused by different G and/or P types. Analyses of serum samples showed that the most frequent G-P combination was G8P6[1], followed by G8P7[5], G8P8[11] and G6P6[1].

Background During the course of development and characterization of various rotavirus reassortants, we found that the relative position of the gene encoding neutralization and protective antigen VP7 of certain rotavirus strains in a PAGE gel was influenced by the concentration of acrylamide. Objectives We investigated systematically various factors that affected the relative position of the rotavirus VP7 gene in a PAGE gel. Study design We analyzed dsRNAs of selected rotavirus strains bearing G1, G2, G3 or G9 specificity by PAGE at varying concentrations of acrylamide. Results We demonstrated that the relative position of the VP7 gene of three G2 strains varied depending upon the concentration of acrylamide in a PAGE gel, which occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus gene background; and the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this phenomenon when the PAGE running conditions were varied. Conclusions The concentration of acrylamide in a PAGE gel was the major factor that influenced the relative position of the VP7 gene of G2 rotavirus strains (i.e., VP7 gene coding assignment by PAGE).

A microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay (PCR-ELISA) has been used for the detection and identification of a variety of microorganisms. Here, we report the development of a PCR-ELISA for the identification of clinically relevant human rotavirus VP7 (G1 to G6, G8 to G10, and G12) and VP4 (P[4], P[6], P[8], P[9], and P[14]) genotypes. The G and P types of reference human and animal rotavirus strains for which specific probes were available were correctly identified by the PCR-ELISA. In addition, reference strains bearing G or P genotypes for which specific probes were unavailable, such as G11, G14, P[3], P[10], and P[11], did not display any cross-reactivity to the probes. The usefulness of the assay was further evaluated by analyzing a total of 396 rotavirus-positive stool samples collected in four countries: Brazil, Ghana, Japan, and the United States. The results of this study showed that the PCR-ELISA was sensitive and easy to perform without the use of any expensive and sophisticated equipment, the reagents used are easy to obtain commercially and advantageous over multiplex PCR since more than one type-specific probe is used and the selection of probes is more flexible.

In this issue of the Journal, the report by Banerjee et al. [1] on the postneonatal protective effect of a prior neonatal infection with G10P [11] rotavirus has important implications for rotavirus vaccinology. The protective efficacy of a neonatal rotavirus infection has been addressed previously by various investigators. In a 1983 Australian study, neonates with asymptomatic rotavirus shedding during the first 14 days of life developed fewer and lesssevere rotavirus-positive diarrhealillnesses postneonatally than did control children [2]. The available strains causing neonatal infection had identical electrophoretic patterns (in addition, 1 strain was identified as an "M-like" [G3] strain [3, 4]), whereas the strains causing postneonatal infection had varying patterns. Although serotypic characterization was not readily available at the time, evidence now indicates that asymptomatic neonatal G3 infections pro

A distinct rotavirus strain (PTRV) was isolated in cell cultures from a stool sample obtained from a diarrheic 3-year-old female pig-tailed macaque (Macaca nemestrina) that was born at the breeding colony of the University of Washington in Seattle. Unlike other known simian rotavirus strains including vervet monkey rotavirus SA11 which bears P5B[2]:G3 or P6[1]:G3 specificity, rhesus monkey rotavirus MMU18006 with P5B[3]:G3 specificity, pig-tailed macaque rotavirus YK-1 with P[3]:G3 specificity and rhesus monkey rotavirus TUCH with P[24]:G3 specificity, the cell-culture-grown PTRV strain was shown to bear P6[1]:G8 specificity as determined by VP4 (P)- and VP7 (G)-specific neutralization assays as well as gene sequence analyses. The virus in the original diarrhea stool was also shown to bear genotypes P[1] and G8. In addition, the PTRV strain exhibited a “long” electropherotype, subgroup I specificity and NSP4 genotype A specificity. The PTRV probe formed (i) 8–9 hybrid bands with genomic RNAs of various bovine rotavirus strains and (ii) only 2–3 hybrid bands with simian rotavirus RNAs as demonstrated by RNA–RNA hybridization, suggesting a possible bovine origin of the virus. Serologic analysis of serum samples obtained from selected pig-tailed macaques in the colony suggested that a rotavirus bearing P[1]:G8 specificity was endemic among macaques for at least 8 years (1987–1994). This is the first report describing an isolation of a simian rotavirus bearing a non-G3 VP7 and possibly a P6[1] specificities. Because of its unique simian serotype, this virus may prove to be valuable in challenge studies in a non-human primate model in studies of rotavirus immunity.

Between May and August in 2003, a total of 251 fecal samples were collected from children and adults with diarrhea (5 inpatients and 246 outpatients) at a private hospital in the city of Ponta Grossa, the state of Paraná, Brazil. Group A rotavirus was detected in 71 of 251 (28.3%) specimens: 55 (77.5%) from children under 5 years of age and 16 (22.5%) from individuals aged 6-72 years. All 71 strains exhibited a "long" RNA pattern when analyzed by PAGE. Sixty-one positive samples that yielded enough RNA were submitted to PCR genotyping. The most frequent G/P genotype combination detected was G1P[8] (86.9%; 53/61) followed by G9P[8] (3.3%; 2/61) and G12P[9] (1.6%; 1/61). Rotaviruses with G2, G3, G4, P[4], or P[6] specificity were not detected. For three strains (4.9%) bearing G1 genotype, the VP4 specificity could no be determined, and two specimens (3.3%) remained G/P non-typeable. One rotavirus strain (HC91) bearing G12P[9] genotype with a "long" electropherotype was isolated from an 11-month-old boy with diarrhea for the first time in Brazil. The cell-culture grown HC91 strain was shown to belong to serotype G12 by neutralization.

There is an urgent need for a rotavirus vaccine, because up to 592,000 infants and young children

Halichonadin G (Ia) and I (II) are found to exhibit cytotoxicity, whereas H (Ib) and J (III) are inactive.

Rotavirus diarrhea is a potentially life-threatening disease that affects millions of children annually around the world. Because protection against rotavirus disease is thought to be type specific, continuous rotavirus surveillance before and after implementation of a vaccine is still of essential importance. Rotavirus surveillance has been conducted in the city of Rio de Janeiro, Brazil since 1982. In the present study, we report rotavirus surveillance data in Rio de Janeiro city from 2000 to 2004. One hundred twenty nine of 1,568 (8.2%) stool samples, collected from children with acute diarrhea between January 2000 and July 2004 were rotavirus-positive. One hundred twenty eight of the 129 (99.2%) rotavirus-positive samples were genotyped for G and/or P specificity. G1 was the predominant strain (49.6%, 64/129) followed by G9 (30.2%, 39/129), and G4 (17.8%, 23/129); G2 and G3 viruses were not detected. One sample (0.8%) was non-typeable. P genotypes were determined for 124 of the 129 (96%) samples, and P[8] was the predominant genotype (90.6%, 117/129). Genotypes P[4] and P[9] were detected in two (1.6%) samples each; one (0.8%) sample presented P[6] genotype; and five (3.8%) samples were non-typeable. Two samples (1.6%) presented mixed P genotypes (P[6] + P[8]). Two unusual strains were isolated: a G8P[4] strain isolated from a non-hospitalized child with diarrhea and a G10P[9] strain isolated from a hospitalized child with diarrhea. J. Med. Virol. 78:263–272, 2006. © 2005 Wiley-Liss, Inc.

Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548–550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744–748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131–141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their “serotypes”. In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.

International audience; Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1,024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.

Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus enterotoxin nonstructural protein (NSP)-4, independent neutralization antigens viral protein (VP)-4 and VP7, and group A rotavirus common antigen VP6 were analyzed by an immunocytochemistry assay in infants who received 1 of several live oral rotavirus vaccines. Significant serum immunoglobulin (Ig) A and IgG antibody responses to homotypic and/or heterotypic NSP4s of genotype [A], [B], or [C] were detected after vaccination. The magnitude of antibody responses to homotypic and heterotypic NSP4s was not significantly different, irrespective of the NSP4 genotype of the administered vaccine strain. In addition, there were no significant differences between IgA antibody responses to homotypic and heterotypic VP7s. In contrast, IgA antibody responses to VP4 were predominantly homotypic. IgA antibody responses to VP7 were lower in magnitude than those to VP4 but were comparable to those to NSP4. Antibody titers to homotypic and/or heterotypic NSP4s were positively correlated with those to VP6 before and after vaccination.

A safe and effective rotavirus vaccine is urgently needed, particularly in developing countries. Critical to vaccine development and implementation is a knowledge base concerning the epidemiology of rotavirus G and P serotypes/genotypes throughout the world. The temporal and geographical distribution of human rotavirus G and P types was reviewed by analysing a total of 45571 strains collected globally from 124 studies reported from 52 countries on five continents published between 1989 and 2004. Four common G types (G1, G2, G3 and G4) in conjunction with P[8] or P[4] represented over 88% of the strains analysed worldwide. In addition, serotype G9 viruses associated with P[8] or P[6] were shown to have emerged as the fourth globally important G type with the relative frequency of 4.1%. When the global G and/or P type distributions were divided into five continents/subcontinents, several characteristic features emerged. For example, the P[8]G1 represented over 70% of rotavirus infections in North America, Europe and Australia, but only about 30% of the infections in South America and Asia, and 23% in Africa. In addition, in Africa (i) the relative frequency of G8 was as high as that of the globally common G3 or G4, (ii) P[6] represented almost one-third of all P types identified and (iii) 27% of the infections were associated with rotavirus strains bearing unusual combinations such as P[6]G8 or P[4]G8. Furthermore, in South America, uncommon G5 virus appeared to increase its epidemiological importance among children with diarrhea. Such findings have (i) confirmed the importance of continued active rotavirus strain surveillance in a variety of geographical settings and (ii) provided important considerations for the development and implementation of an effective rotavirus vaccine (e.g. a geographical P-G type adjustment in the formulation of next generation multivalent vaccines).

One hundred fifty-seven (23%; n = 678) rotavirus-positive stool samples were collected between March 1997 and December 1999 in the cites of Rio de Janeiro and Niterói. Rotaviruses in 143 (91%) samples were genotyped by reverse transcription-PCR for G and/or P specificity. Rotaviruses in the majority of G-P-typeable samples (73.3%; 74 of 101) were identified as having globally common genotypes G1P[8], G2P[4], G3P[8], and G4P[8]. Unusual strains such as G1P[9], G2[P8], G3P[9], and G9P[4] strains were detected in 8.9% (9 of 101) of the samples. Genotypes G9P[8], G9P[6], and a mixture of G9 and other G or P types represented 15.9% (25 of 157) of the isolates. Mixed infections were detected in 25 (15.9%) samples, and rotaviruses in 15 samples (9.6%) were not typed.

Rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide. Surveillance of rotavirus serotypes/genotypes (both VP7[G] and VP4[P]) is in progress globally in which polymerase chain reaction (PCR) has been the assay of choice. We investigated polymorphism of the VP7 gene of serotype G9 rotavirus strains and its impact on the determination of VP7 gene genotype by PCR assay. By VP7 gene sequence analysis, we and others have previously shown that the G9 rotavirus strains belong to one of three VP7 gene lineages. By PCR assay using three different sets of commonly used primers specific for G1-4, 8 and 9, 23 Brazilian G9 strains and 5 well-characterized prototype G9 strains which collectively represented all three VP7 gene lineages were typed as: (i). G3; (ii). G4; (iii). G9; (iv). G3 and G9; or (v). G9 and G4 depending on a primer pool employed. This phenomenon appeared to be due to: (i). a VP7 gene lineage-specific polymorphism, more specifically mutation(s) in the primer binding region of the VP7 gene of G9 strain; and (ii). the magnitude of difference in nucleotide homology at respective primer binding site between homotypic (G9) and heterotypic (G3 or G4) primers present in a primer pool employed.

Rotaviruses are the single most important etiologic agents of severe diarrhea in infants and young children worldwide. They possess a triple capsid morphology and a genome of 11 segments of double-stranded (ds) RNA. During the course of the development of various live, attenuated reassortant rotavirus vaccines, we often experienced difficulty in identifying the parental origin of certain genome segment(s) of a reassortant vaccine candidate. Various assays have been utilized for determination of the parental origin of reassortant virus genes, including polyacrylamide gel electrophoresis (PAGE), DNA and/or RNA hybridization assays and gene sequence analysis. The traditional PAGE is simple and easy to perform, however, it is common to find that certain cognate dsRNA segment(s) cannot be differentiated by this assay due to a high degree of sequence homology among different rotavirus strains. Constant denaturant gel electrophoresis (CDGE) is one of several methods that have been used to screen DNA fragments for small sequence changes or point mutations. By using the CDGE, we were successful in partially denaturing rotavirus dsRNA thereby changing the physical properties of the genome segment(s) in the gel and thus differentiating the cognate genome segment(s) of rotavirus reassortants. The CDGE provides a simple and reliable assay system for identification of parental gene origins of a rotavirus reassortant.

During a phase 3 clinical trial of rhesus monkey rotavirus-based quadrivalent vaccine in Venezuela, 2207 infants received 3 oral doses of vaccine (4 x 105 plaque-forming units/dose) or placebo at ages approximately 2, 3, and 4 months; 219 (14%) of 1537 stools obtained during 1550 diarrheal episodes in postvaccination surveillance were rotavirus-positive by enzyme-linked immunosorbent assay. With the use of various VP7 and VP4 primers for genotyping purposes, 213 of 219 rotavirus-positive stools were analyzed by reverse-transcription polymerase chain reaction. Twenty-nine (14%) of 213 rotavirus-positive stools contained at least 2 distinct rotavirus strains: a low-titered vaccine strain(s) and a second strain that, when possible, was studied further and found to be a wild-type rotavirus strain. The titer of vaccine viruses in 19 stools that plaqued directly in cell cultures ranged from 10(1) to 10(3) plaque-forming units/0.5 mL of a 10% stool suspension. Reassortants of vaccine virus and wild-type human rotavirus were not detected.

The global disease burden of rotavirus diarrhea in infants and young children has stimulated interest in the biological and clinical characteristics of these agents, leading to intensive efforts to develop a vaccine. A rhesus rotavirus (RRV)-based quadrivalent vaccine ("RotaShield") was licensed and administered to about 1 million infants and found to be highly effective. However, it was withdrawn because of a link with intussusception. This vaccine was developed according to a modified "Jennerian" approach in which one of the two major outer capsid proteins (VP7) shares neutralization specificity with one of the four epidemiologically important human rotavirus serotypes. The other outer capsid protein (VP4) is derived solely from RRV and is distinct from the VP4 of the four human rotavirus serotypes of epidemiologic importance. In an effort to further increase the immunogenicity of the existing VP7-based RRV quadrivalent vaccine, we generated three single VP4 gene substitution reassortant rotavirus candidate vaccines, each of which bears a single human rotavirus VP4 gene encoding P serotype 1A[8] or 1B[4] specificity while the remaining 10 genes are derived from the rhesus rotavirus. By incorporating one or two of these strains into the quadrivalent vaccine, a pentavalent or hexavalent RRV-based vaccine could be formulated thus providing antigenic coverage not only for VP7 serotype 1, 2, 3 and 4 but also for VP4 serotype 1A[8] or 1B[4], thus possibly augmenting its immunogenicity. Similarly, three single VP4 gene (P1A[8] or P1B[4]) substitution reassortants have also been generated in a background of 10 bovine (UK) rotavirus genes for addition to a second generation UK-based quadrivalent vaccine.