Spindle-shaped viruses are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G+C (approximately 40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCRamplified 16S rRNA genes and by cultivation. High viable counts (10 6 CFU/ml) were obtained on solid media. Long incubation times (>8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids.Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response.Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought.Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly.

Antigenic sites on the rotavirus major outershell glycoprotein were identified by using mutant viruses selected for resistance to neutralizing (serotype-specific) monoclonal antibodies. The glycoprotein genes from these mutants were sequenced to determine the position and nature of the resultant amino acid substitutions in the protein. Three regions (A, B, and C) were identified (amino acids 87-96, 145-150, and 211-223, respectively), of which region C appears to be the most important. A mutation in region C caused a 10-fold increase in resistance to neutralization by polyclonal antiviral antiserum. The results of this study, together with other data, indicate that the three-dimensional folding of the native protein is such that regions A and C are in close proximity.Diarrheal disease remains one of the major health problems in developing countries and is responsible for a large number of deaths each year, particularly among infants (1). Rotavirus has been identified as the single most important causative agent of acute infantile gastroenteritis (2), and a worldwide effort is being made to develop a suitable rotavirus vaccine (3, 4). Rotaviruses are 70-nm-diameter spherical particles with segmented double-stranded RNA genomes, and four human serotypes are known (5, 6). Protection from disease appears to be effected by secretory antibodies directed against determinants on the two recognized surface proteins (one the product of gene 4, and the other, of genes 7, 8, and 9) (7). The most important of these is the major outer-shell glycoprotein (8), which determines the virus serotype (9-11). Recent work has focused on this protein and a considerable amount of information is now known, including its amino acid sequence (12-15) and its overall antigenic structure (16). However, analysis of the antigenic structure at the amino acid level has not been reported. We describe here the identification of type-specific antigenic determinants on the major outer-shell glycoprotein of SA11 rotavirus by the characterization of mutants resistant to neutralizing monoclonal antibodies. MATERIALS AND METHODSVirus. SA11 rotavirus was grown in MA104 cells as described (17). To produce stocks for the selection of mutant viruses, a single batch of virus, which had not previously been plaque-purified, was passaged once at very low multiplicity (-5 x 10-4 plaque-forming unit per cell) and the harvests from each culture bottle were stored at -70°C as separately numbered stocks.Selection of Monoclonal Antibody (mAb)-Resistant Mutants. The mAbs used for the selection of viral mutants have been described (16,18). The methods used for selecting and plaque-purifying mAb-resistant mutants will be described in detail elsewhere.Production of Virus Transcripts. Virus was purified by centrifugation in CsCl gradients as described (17). After treatment with EDTA to activate the viral polymerase (19), virus cores were incubated in 100 mM Tris Cl buffer (pH 8.0, 500 suI) containing 10 mM MgCl2, 100 mM sodium acetate, 2 mM ATP, 2 mM GTP, 2 mM CT...

Strains C23T and HBSQ001 were isolated from solar salterns and are novel square-shaped, aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media containing 18 % salts at around neutral pH. Mg2+ is not required. The DNA G+C content of both isolates was 46.9 mol% and DNA–DNA cross-hybridization showed a relatedness of 80 %. Their 16S rRNA gene sequences showed only 2 nucleotide differences (99.9 % identity) and phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halogeometricum borinquense PR 3T (91.2 % sequence identity). The major polar glycolipid of both isolates was the sulfated diglycosyl diether lipid S-DGD-1. Electron cryomicrosopy of whole cells revealed similar internal structures, such as gas vesicles and polyhydroxyalkanoate granules, but the cell wall of isolate HBSQ001 displayed a more complex S-layer compared with that of isolate C23T. The phenotypic characterization and phylogenetic data support the placement of isolates C23T and HBSQ001 in a novel species in a new genus within the Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov., sp. nov. The type strain of Haloquadratum walsbyi is C23T (=JCM 12705T=DSM 16854T).