Novel hemotropic mycoplasmas are widespread and genetically diverse in vampire bats.

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

[An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes].

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

Genetic diversity of hantaviruses associated with hemorrhagic fever with renal syndrome in the far east of Russia.

To identify the hantaviruses causing hemorrhagic fever with renal syndrome (HFRS) in the Far East of Russia, blood samples collected from HFRS patients in 1994-1998, were examined by reverse transcription-polymerase chain reaction. In addition, 36 sera were tested by an immunofluorescence assay for antibodies against Hantaan, Seoul, Puumala, and Khabarovsk viruses, and 54 samples were tested by plaque reduction neutralization test. With both serological assays, the highest antibody titers were to Hantaan and/or Seoul viruses. Of 110 blood samples 36 were found RT-PCR positive. Phylogenetic analysis the sequences of a 256-nucleotide (nt) fragment of the hantavirus M genome segment revealed at least 3 genetically distinct hantavirus lineages. Nucleotide sequence comparison showed that two of the lineages, designated as FE and Amur (AMR), differed from one another by 15.9-21.2% and from Hantaan virus by 9.8-17.5%. The third lineage, VDV, differed from Seoul virus by 2.6-5.1%. All S segment sequences were from FE lineage, and differed from Hantaan virus by 10.7-12.6%. Thirty of the 36 (83%) analyzed sequences were found to be the FE genotype, which is very similar to that of Hantaan virus, strain 76-118. Of the remaining hantaviruses, 11% were the AMR genotype, and 6% the VDV genotype, which are genetically novel genotypes of Hantaan or Seoul viruses, respectively.

Alastrim smallpox variola minor virus genome DNA sequences.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.

Cloning and characterization of the gene encoding M.FauI DNA methyltransferase.

The enzymes of the FauI restriction-modification system from the Flavobacterium aquatile strain recognize the non-palindromic sequence 5'-CCCGC-3'/3'-GG-GCG-5'. We have cloned the gene encoding the DNA modifying component of this system and determined its nucleotide sequence. The deduced amino acid sequence contains ten conserved motifs characteristic for [cytosine-5] DNA methyltransferases. Part of the gene sequence that encodes the putative target recognizing domain of the M.FauI shows some homology with the downstream region, thus indicating that duplication of the DNA segment was probably involved in the gene evolution.

Terminal region sequence variations in variola virus DNA.

Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.

Complete genetic characterization and analysis of isolation of Sin Nombre virus.

This study reports completion of the genetic characterization of the entire genome of Sin Nombre (SN) virus (NMH10) detected in autopsy tissues from a patient who died of hantavirus pulmonary syndrome (HPS). The large (L) genome segment was found to be 6,562 nucleotides in length and encoded a putative L polymerase that was 2,153 amino acids in length. No evidence of segment reassortment with other well-characterized hantaviruses was obtained. The sequence of the entire S, M, and L genome segments of SN virus (strain NMR11) isolated from a mouse (trapped in the residence of the patient infected with SN virus [NMH10]) by passage two times in Peromyscus maniculatus and then by five passages in E6 Vero cells was determined and compared with that of the virus detected in autopsy tissues. Only 16 nucleotide differences were detected between the virus genomes, and none of these resulted in virus protein amino acid substitutions. Determination of the exact 5'- and 3'-terminal sequences of all genome segments of SN virus and representatives of other serologic groups in the Hantavirus genus, family Bunyaviridae, showed the existence of conserved nucleotide domains that may be involved in important regulatory mechanisms, such as RNA encapsidation, polymerase binding, and control of transcription and replication.

[Study of the structure-function organization of the variola virus genome. IV. Sequencing and analysis of the nucleotide sequence of the right terminus of the India-1967 strain genome]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. IV. Sekvenirovanie i analiz posledovatel'nosti nukleotidov pravogo kontsa genoma shtamma Indiia-1967.

Sequencing and computer analysis of the variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region have been carried out. Fifty nine potential open reading frames (ORFs) of over 60 amino acid residues have been identified. Structure-function organization of VAR-IND DNA segment under study was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple distinctions in the genetic map of VAR-IND from VAC-COP and VAC-WR have been revealed along with the high similarity to the corresponding VAR-HAR segment. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

[The use of a biotinated probe for the detection of the genomic RNA of the hepatitis A virus in clinical specimens]. / Ispol'zovanie biotinirovannogo zonda dlia detektsii genomnoi RNK virusa gepatita A v klinicheskikh obraztsakh.

A detection technique for hepatitis A virus (HAV) RNA by means of molecular hybridization using ss-biotinated DNA probe on the basis of M13 bacteriophage is described. The technique sensitivity reached 1-10 pg of control DNA or 100-500 pg of HAV. The experiments for detection of HAV carriers among the patients and contacts from foci of HAV outbreaks were carried out. A comparative analysis of the above technique and enzyme immunoassay and amplification technique was done and good coincidence of the results was demonstrated.

[Structure-activity organization of the variola virus genome. III. Sequencing and analysis of the nucleotide sequence of the conserved region of HindIII-F, -N-, and -A-fragments of the India 1967 strain genome]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. III. Sekvenirovanie i analiz posledovatel'nosti nukleotidov konservativnogo raiona HindIII-F-, -N-, i -A-fragmentov genoma shtamma Indiia-1967.

Computer analysis of variola major virus (VAR) genomic fragment bounded by open reading frames (ORFs) D1R and A33L which is 47,961 bp long revealed 46 potential ORFs. The VAR proteins were compared with the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion body protein. The possible functions of the analyzed viral proteins are discussed.

[Study of the structure-activity organization of the variola virus genome. II. Analysis of the nucleotide sequence of the HindIII region (C,E,R,Q,K and H)-DNA fragments of the India-1967 strain]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. II. Analiz posledovatel'nosti nukleotidov raiona HindIII (C,E,R,Q,K i H)-fragmentov DNK shtamma india-1967.

Sequencing of variola virus (VAR) genome region of 43069 bp was carried out. This area contains 42 potential genes. Computer analysis of proteins coding for these viral genes was done. We compared VAR proteins with the those of vaccinia virus. The region studied is conservative for orthopoxviruses.

Nucleotide sequence of XhoI O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.

The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions.

[New type II and IIs restriction endonucleases from thermophilic bacteria of the genus Bacillus]. / Novye éndonukleazy restriktsii tipa II i IIs iz termofil'nykh bakterii roda Bacillus.

Restriction endonucleases have been isolated from 26 strains of thermophilic strains of the Bacillus genus, their recognition sequences were determined, and for 15 of them cleavage sites identified. The enzymes proved to be isoschizomers of known endonucleases BstNI, EarI, HaeIII, HpaII, Cfr10I, BsiYI, BclI, BbvII, BbvI, BstEII, BsaBI, BsrI, FspI, ClaI, SfeI.

[The isolation and study of the characteristics of a cytopathic strain of the hepatitis A virus]. / Vydelenie i izuchenie kharakteristik tsitopaticheskogo shtamma virusa gepatita A.

A fast-growing cytopathic isolate of human hepatitis A virus (strain MB-7) was derived from fecal samples of infected patients and adapted to growth in FRhK-4 cell culture. A positive serum standard against HAV and electron microscopy were used to demonstrate that MB-7 belonged to human hepatitis A virus. The strain MB-7 induced plaque formation in FRhK-4 under agar overlay after 10-12 days of incubation. The PCR products of gene VP1 were cloned in E. coli and its primary structure was determined. MB-7 was shown to have more homology with HAV strains isolated in the USA and China.

Nucleotide sequence analysis of variola virus HindIII M, L, I genome fragments.

DNA of the variola major virus strain India-1967 in the region of HindIII M, L, I fragments has been sequenced. Analysis of this sequence of 18029 bp revealed 19 potential open reading frames (ORFs). Four proposed proteins (L2R, H9R, L5L, L6R) contain metal-binding domains. Comparison of the variola virus (VAR) and vaccinia virus strain Copenhagen (COP) sequences show that the main differences are between proteins L1R and I5R. L1R contains 6 additional amino acid residues on the C-terminus. The protein I5R of VAR contains three Ca2+ binding domains but this COP has deletions in 2 of the 3 established domains. Possible functions of the predicted viral polypeptides are discussed.

[Study of the structural-functional organization of the natural variola virus genome. I. Cloning HindIII- and XhoI-fragments of viral DNA and sequencing HindIII -M, -L, -I fragments]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. I. Klonirovanie HindIII- i XhoI-fragmentov virusnoi DNK i sekvenirovanie HindIII-M, -L, -I fragmentov.

HindIII and XhoI genome fragments of variola major virus strain India-1967 were inserted into the bacterial plasmids and cosmid. Sequencing and computer analysis of the region of HindIII M, L, and I DNA fragments of the virus studied have been carried out.

[Restriction endonucleases: the detection of new producer strains and the isolation of enzyme preparations]. / Endonukleazy restriktsii: vyiavlenie novykh shtammov produtsentov i poluchenie fermentnykh preparatov.

The method for analysis of microorganisms for the presence of the modification-restriction systems has been developed. The method has permitted to detect more than 10 new producing strains of restrictases including microorganisms of Rhizobium genus. Some of them are promising for practical use. It has been shown that using selection of clones the strain productivity can be increased. The purification process for the majority of restrictases has been proposed. Some physical and catalytic properties of new enzymes have been studied.

[Molecular biological study of the vaccinia virus genome. IV. The late nonstructural 36K protein of vaccinia virus is vitally important]. / Molekuliarno-biologicheskoe izuchenie genoma virusa ospovaktsiny. IV Gen pozdnego nestrukturnogo belka 36K virusa ospovaktsiny iavliaetsia zhiznenno vazhnym.

Two genetics markers: the herpes simplex virus thymidine kinase and Escherichia coli beta-galactosidase genes were inserted into the 36K protein gene of vaccinia virus located in a HindIII-P DNA fragment. An unstability of recombinant viruses with Lac(+)-phenotype were discovered. A mechanism of viruses unstable variants formation was proposed, it was confirmed by the results of hybridisation analyses of virus recombinant genomes. The importance of a late nonstructural 36K protein gene for virus reproduction was demonstrated.