Molecular and immunologic characterization of group A bovine rotavirus field isolates with P8[11] spike protein

Bovine rotavirus strain B223 is the North American prototype for group A P8[11] serotype/genotype rotaviruses. Rotaviruses with this serotype/genotype are a prevalent cause of neonatal calf diarrhea and have also been isolated from asymptomatic human infants. On the basis of deduced amino acid sequence of the outer capsid viral protein 4 (VP4), strain B223 lacks a cysteine at position 318 that is conserved among all other rotavirus strains. It has been speculated that this may result in the loss of disulfide bond and a change in the structure of the VP4 that may affect the infectivity and antigenicity of the virus. This paper describes partial sequences of the VP4 gene (nucleotides 613 to 1016 coding for amino acid positions 202 to 335) of 16 bovine rotavirus field isolates with P8[11] that were obtained from calves in Nebraska and Indiana. All the isolates lack a cystein at position 203 and had a conserved valine residue at position 318, thus indicating that the prototype strain B223 is representative of group A rotaviruses with P8[11] serotype/genotype.     

Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain

The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1-4, and 6-11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype.     

Toxoplasmosis infection in pregnant women in Lanzhou

A strain of poliovirus type 1 isolated from an endemic in Shandong Province in 1988 and three strains of poliovirus type 1 isolated from an endemic in Xinjiang in 1990 were analysed by dot blot hybridization with Sabin I specific probe and PCR amplification with Sabin I specific primers. They were proved to be non-Sabin-like poliovirus. The nucleotide sequences of VP1-2A junction region of four poliovirus type 1 isolates were analysed. It was found that the nucleotide diversity of four isolates with Sabin I was equal to or greater than 16.7%. This finding proved that they were wild polioviruses and greatly different fromSabin I. It was also found that the nucleotide sequence diversity among three strains from Xinjiang was very small (less than 2.6%) with identical amino acid sequences. This indicated that the three Xinjiang strains belonged to the same genotype. While the difference of Shandong strain from the three Xinjiang strains was between 14. 0% to 15.33% in nucleotide sequence and 2.0% to 4.0% in amino acids. This indicated that Shandong strain was apparently different from three Xinjiang strains and it belonged to another wild genotype which had a different history of evolution.     

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.     

Molecular epidemiological analysis of Pseudomonas aeruginosa by pulsed-field gel electrophoresis]

In order to see the nosocomial infection by Pseudomonas aeruginosa (P. aeruginosa) in the Akita University Hospital, we analyzed the serotype and genotype for 150 strains isolated from various clinical specimens from 1994 to 1996. One hundred strains chosen at random were divided into 11 serotypes by serotyping and into 50 genotypes by pulsed-field gel electrophoresis of Spe I-digested P. aeruginosa genomic DNAs. Serotype E strains, most common, gave 17 genome patterns. Fifty strains separated periodically in certain wards had further 7 genome patterns. The strains isolated from one patient with different times or with different serotypes showed the same stable genotype. Genome patterns were inconsistent with susceptibility to antimicrobial agents. Both drug-susceptible and -resistant strains were found in strains with the same genome pattern. The genome pattern was identical, even though the susceptibility was different due to isolation time or storage. This suggested that the multidrug-resistant strains of P. aeruginosa expanded in the hospital were not derived from one original strain.     

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus

Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E. coli.     

Evaluation of multiple Pseudomonas aeruginosa strains with different characteristics in clinical specimens; combined results from serotyping, drug susceptibility and enzyme production

Multiple Pseudomonas aeruginosa strains with different characteristics are occasionally isolated from a clinical specimen. Therefore, more than five isolated colonies of P. aeruginosa obtained at random from each clinical specimen (47 sputa, 18 urine, 10 pus and 8 others). These were investigated for serotype, drug susceptibility to eight antimicrobial agents and productivity of enzymes, such as protease and elastase. The specimens with multiple serotype colonies were shown in 17% of the sputa, 11% of the urine and 10% of the pus. 45.7% of the specimens with single serotype colonies exhibited more than two different patterns of enzyme productivity and so did 47.1% different patterns of drug susceptibility. Single serotype strains of P. aeruginosa with different characteristics of these tests were demonstrated in 81.3% of the urine, 73.6% of the sputum, 50.0% of the pus and 66.7% of others. We conclude that it is important to recognize the possible existence of multiple P. aeruginosa strains with different patterns of the enzyme productivity and drug susceptibility, regardless of single serotype, in clinical specimens.     

Hypertension, diuretics, and antihypertensive medications as possible risk factors for renal cell cancer

We identified the serotypes and genomes patterns of 100 strains of Pseudomonas aeruginosa (P. aeruginosa) that had been isolated from patients who were admitted to the hospital of the Fukui Medical School between 1992 and 1995. A monoclonal diagnostic kit was used to identify the serotypes. Genome patterns were determined by pulsed field gel electrophoresis (PFGE). Serotypes A, B, C, D, E, F, G and I exhibited distinct genome patterns. Differences in genome patterns were also observed in strains of serotypes E and G, depending on the types of clinical samples collected and/or the area of the hospital from which they were isolated. Many of the multiple antibiotic-resistant strains of P. aeruginosa exhibited serotype E. The genome pattern differed between strains that were susceptible vs. resistant to multiple antibiotics. The latter strains exhibited similar genome patterns regardless of their origin. These findings suggest that analysis of genome patterns is important for identifying the origin of nosocomial infection caused by P. aeruginosa, serotype E.     

Restriction endonuclease analysis of plasmid DNA of Yersinia pseudotuberculosis infections in Shimane Prefecture, Japan

The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species.     

Electrophoretic typing of nosocomial rotavirus infection in a general paediatric unit showing the continual introduction of community strains

During 1989 stool specimens from hospitalised children with gastroenteritis at Ga-Rankuwa Hospital in South Africa were examined for the presence of rotaviruses. Overall 16% of the children were positive for rotavirus. However, 43% of the rotavirus positive patients were infected in the hospital. Further characterisation of the rotavirus strains was performed by electrophoresis of the RNA genome and hybridisation analysis of the VP7 and VP4 genes present. The strains associated with nosocomial infection were similar to those strains acquired in the community. The majority of the strains, both community- or hospital-acquired, were associated with a serotype 1 strain with a long electrophoretype and bearing the Wa-like VP4 gene. Three minor rotavirus strains with a long electrophoretype were also observed to be circulating bearing serotype 1 or 4 VP7 genes and the Wa-like VP4 gene. Interestingly, a serotype 4 strain bearing the M37-like VP4 gene was identified to occur almost exclusively in neonates although the gene was associated with diarrhoea in these cases. Two strains with differing short RNA electrophoretypes were also observed, members of which hybridised to VP7 serotype 2 and VP4 DS-1 type probes.     

Somatostatin-14, somatostatin-28, and prosomatostatin[1-10] are independently and efficiently processed from prosomatostatin in the constitutive secretory pathway in islet somatostatin tumor cells (1027B2)

Seven neutralizing monoclonal antibodies (nMAbs) produced against serotype Asia-1 foot-and-mouth disease virus (FMDV) were used to select neutralization-resistant variants. Seven single and six multiple antibody-resistant variants were selected to identify neutralization antigenic sites on FMDV Asia-1. The variants no longer reacted with nMAbs which were used to select them when tested by microneutralization test (MNT), radioimmunoassay (RIA) and agar gel immunodiffusion (AGID) assay. Based on the binding and neutralization patterns of the variants, the nMAbs could be divided into discrete groups indicating the presence of three independent antigenic sites with evidence for occurrence of possibly a fourth site on the virus surface. Site 1 was present on 140S, 12Sps and VP1 and thus was conformation-independent. Sites 2 and 3 were restricted to the intact virion (140S) and thus were more conformation-dependent. Site 4 present on 140S virions and 12S protein subunits was less conformation-dependent. The site 3 nMAbs neutralized the infectivity of all the ten different Asia-1 virus isolates tested indicating that this site is conserved in Asia-1 virus serotype. Both cross-neutralization of different Asia-1 viruses with the nMAbs and cross-inhibition assays between MAbs demonstrated that the nMAbs recognized at least six different epitopes on Asia-1 virus.     

G (VP7) serotype-dependent preferential VP7 gene selection detected in the genetic background of simian rotavirus SA11

We previously found the preferential selection of VP7 gene from a parent rotavirus strain SA11 with G serotype 3 (G3) in the sequential passages after mixed infection of simian rotavirus SA11 and SA11-human rotavirus single-VP7 gene-substitution reassortants with G1, G2, or G4 specificity. However, it has not been known whether or not VP7 genes derived from other strains with G3 specificity (G3-VP7 gene) are preferentially selected in the genetic background of SA11. To address this question, mixed infections followed by multiple passages were performed with a reassortant SA11-L2/KU-R1 (SKR1) (which possesses VP7 gene derived from G1 human rotavirus KU and other 10 genes of SA11 origin) and one of the five G3-rotaviruses, RRV, K9, YO, AK35, and S3. After the 10th passage, selection rates of SA11-L2/KU-R1 gene 9 (G1-VP7 gene) and gene 5 (NSP1 gene) reduced considerably (0 to 20.4%) in the clones obtained from all the coinfection experiments, while all or some of other segments were preferentially selected from SKR1 depending on the pairs of coinfection. When viral growth kinetics was examined, SKR1 exhibited better growth and reached a higher titer than any G3 viruses. Although the generated reassortants with VP7 gene and NSP1 gene derived from G3 viruses showed almost similar growth kinetics to that of SKR1 during the first 20 h of replication, the titers of these reassortants were higher than that of SKR1 after 36 h postinfection. The results obtained in this study suggested that G3-VP7 gene is functionally more adapted to the genetic background of SA11.     

Immunological study of Japanese encephalitis virus--serological analysis of strains isolated from Thailand, India, Singapore and Taiwan

Antigenic comparison of the twenty-two Japanese encephalitis (JE) virus strains, which were isolated from Taiwan, Singapore, Thailand and India between 1963 and 1984, was carried out by the hemagglutination inhibition (HI) test using the 15 monoclonal antibodies characterized by the different reactivities against Nakayama-RFVL, Beijing 1, Kamiyama, Muar or 691004 strain. Of these twenty-two strains, the seventeen strains reacted with the Kamiyama type-specific monoclonal antibody (KAMIMA 6), but anti-Nakayama, anti-Beijing 1 and anti-Muar type-specific antibodies showed no reactivities with any of the strains. This suggested that the currently prevalent JE virus strains in these areas belonged to the Kamiyama serotype. The other five strains (ThCMP 1982, KE083, KE093, 733913 and Ling) did not react with the above four type-specific monoclonal antibodies. Of these five strains, however, all except the KE093 strain showed a similar pattern to the Kamiyama strain on the basis of the HI reactivities against the other eleven antibodies. The KE093 isolated from Thailand in 1983 showed immunologically outstanding different from the other strains. This result showed the immunological diversity of the JE virus.     

Nucleotide sequence of the VP4-encoding gene of an unusual human rotavirus (HCR3)

Human rotavirus strain HCR3 was isolated from the stool of a clinically normal infant and identified as a serotype G3 rotavirus; however, it could not be grouped into any known human VP4 genetic groups by a polymerase chain reaction assay. The fourth gene of strain HCR3, which encodes the outer capsid protein VP4, was sequenced. This gene is 2362 nucleotides in length and contains one open reading frame capable of encoding a protein of 776 amino acids. The VP4 protein of strain HCR3 shared 67.5-73.5% amino acid identity with those of strains KU, RV-5, 1076, and K8, representing four human genetic groups, and relatively high homology (84.7%) with a fifth genetic group represented by strain 69M, whose VP4 shows more similarity to animal than to human strains. Strain HCR3 shared higher VP4 amino acid homology with various animal rotaviruses, ranging from 74.5 to 89.4%. These observations suggest that the VP4 outer capsid protein of strain HCR3 represents a new VP4 genetic group that is more closely related to animal rotaviruses than to human rotaviruses.