Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Recombination among Protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the Protein II family

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.     

Far‐eastern strains of bean common mosaic virus and methods of their immunodiagnostics

The paper presents data of investigation on the physico‐chemical and antigenic properties of capsid proteins of the Bean common mosaic virus isolated from Phaseolus plants in the Russian Far East (BCMV‐R) and from China (BCMV‐C). A method for isolation of the virus preparation was selected. The purified preparations of two isolates BCMV have been obtained. The presence of one polypeptide in structural proteins of virions was established and their molecular masses determined (BCMV‐R ‐ 31,6 kD; BCMV‐C ‐ 32,1 kD). Polyclonal antiserum was obtained with titre 1:12800 and the indirect and “sandwich"‐variants of ELISA were developed to detect this virus. The allied relationships were established with the bean yellow mosaic virus and with the type representative of the genus Potyvirus ‐ PVY. Based on the data of physico‐chemical and antigenic properties it was concluded that isolates BCMV‐R and BCMV‐C are two independent strains of this virus. The presence of strain‐, virus‐ and genusspecific epitopes of capsid proteine was revealed as a result of comparison of antigenic characteristics of the Russian Far Eastern and Chinese strains of BCMV. A high antigenic activity of capsid protein of the Russian Far Eastern strain was observed.     

Antigenic analysis of Methanomicrobiales and Methanobrevibacter arboriphilus.

Preparation of new antisera has permitted more comprehensive immunological analyses of the two families of Methanomicrobiales. Methanomicrobiaceae and Methanosarcinaceae, and the species Methanobrevibacter arboriphilus. Immunological analysis was carried out with antibody probes against 23 strains, including almost all genera and species of methanogens. The absence of cross-reactions between families of methanogens was confirmed. Methanomicrobium and Methanogenium were found to be immunologically related. Extensive cross-reactions occurred among six strains of Methanosarcinaceae, but none occurred among three strains of M. arboriphilus when tested with the S probe, i.e., the last antiserum dilution of the titration curve's plateau.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.     

Study of immunochemical heterogeneity of Azospirillum brasilense lipopolysaccharides

A comparative immunochemical analysis of lipopolysaccharides (LPS) in Azospirillum brasilense model strains Sp7 and Sp245 and in mutants with transformed somatic antigens has been performed. According to the results of a complex of various immunochemical methods, including studies with polyclonal antibodies against the LPS these bacteria, their LPS consist of an assembly of macromolecules with different antigenic characteristics. Two types of O-specific polysaccharides (O-PS) are present in the LPS of every strain of A. brasilense under study. The major difference between the two O-PS is the antigenic heterogeneity of one of them. This heterogeneous O-PS has been shown to possess at least two O-factors (antigenic determinants) different in their structure. Meanwhile, according to all the tests performed, the other O-PS in every strain is immunochemically homogeneous and identical to one of the determinants revealed in the more diversified O-PS. The LPS heterogeneity among the given strains may be due to the pattern of O-specific polysaccharide synthesis, one of the O-PS being an intermediate in the synthesis of the other.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

Engineering a poliovirus type 2 antigenic site on a type 1 capsid results in a chimaeric virus which is neurovirulent for mice.

Poliovirus type 2 (PV-2) Lansing strain produces a fatal paralytic disease in mice after intracerebral injection, whereas poliovirus type 1 (PV-1) Mahoney strain causes disease only in primates. Atomic models derived from the three-dimensional crystal structure of the PV-1 Mahoney strain have been used to locate three antigenic sites on the surface of the virion. We report here the construction of type 1-type 2 chimaeric polioviruses in which antigenic site 1 from the PV-1 Mahoney strain was substituted by that of the PV-2 Lansing strain by nucleotide cassette exchange in a cloned PV-1 cDNA molecule. These chimaeras proved to have mosaic capsids with composite type 1 and type 2 antigenicity, and induced a neutralizing response against both PV-1 and PV-2 when injected into rabbits. Moreover, a six-amino-acid change in PV-1 antigenic site 1 was shown to be responsible for a remarkable host-range mutation in so far as one of the two type 1-type 2 chimaera was highly neurovirulent for mice.     

A comparison of some properties of four strains of cherry leaf roll virus

The elm mosaic and golden elderberry strains of cherry leaf roll virus (CLRV) and a strain from cherry and from rhubarb were very similar in their host range, symptomatology and properties in vitro. However, only the rhubarb isolate infected rhubarb systemically and only the golden elderberry isolate infected Sambucus nigra systemically. Purified preparations of all strains contained isometric particles which sedimented as two nucleoprotein components with sedimentation coefficients of about 115 S and 128 S. The elm mosaic strain was the least stable in vitro and was the most difficult to purify. In plant-protection tests, one-way protection occurred between tomato ringspot virus and each of the four CLRV strains. However, whereas the elm mosaic, golden elderberry and the cherry strains protected against one another, they did not protect against infection with the rhubarb strain.     

Study of immunochemical heterogeneity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> lipopolysaccharides

A comparative immunochemical analysis of lipopolysaccharides (LPS) in Azospirillum brasilense model strains Sp7 and Sp245 and in mutants with altered somatic antigens has been performed. According to the results of a complex of various immunochemical methods, including studies with polyclonal antibodies against the LPS these bacteria, their LPS consist of an assembly of macromolecules with different antigenic characteristics. Two types of O-specific polysaccharides (O-PS) are present in the LPS of every strain of A. brasilense under study. The major difference between the two O-PS is the antigenic heterogeneity of one of them. This heterogeneous O-PS has been shown to possess at least two O-factors (antigenic determinants) different in their structure. Meanwhile, according to all the tests performed, the other O-PS in every strain is immunochemically homogeneous and identical to one of the determinants revealed in the more diversified O-PS. The LPS heterogeneity among the given strains may be due to the pattern of O-specific polysaccharide synthesis, one of the O-PS being an intermediate in the synthesis of the other.     

Antibody analysis of relationships among methanogenic bacteria

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.     

Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria.

The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells.     

Human immunodeficiency virus type 1 intersubtype (B/E) recombination in a superinfected chimpanzee.

Genetic characterization of a large number of human immunodeficiency virus type 1 (HIV-1) isolates indicates that at least 10% of all strains have mosaic genomes generated by recombination between viruses of the same or different subtypes or clades. What is not known, however, is the time between infection with the first and second HIV-1 strains as well as the time between infection with the second strain and the recombinational event. After 32 months of infection with HIV-1(LAI(IIIB)), a chimpanzee was inoculated intravenously and became infected with a subtype E strain, HIV-1(90CR402). With PCR amplification, DNA heteroduplex analysis, and DNA sequencing, both parental strains and two distinct recombinant proviruses were found in genomic DNA from lymph node tissue obtained 24 weeks after exposure to HIV-1(90CR402). These results show (i) that antiviral immune responses established by long-term infection with an HIV-1 subtype B strain did not prevent infection by a subtype E strain and (ii) that both strains actively replicated and produced sufficient quantities of virus to coinfect the same cell(s), resulting in recombinant viruses.