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.

Variations in genome fragments coding for RNA polymerase in human and simian hepatitis A viruses.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.

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.

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.

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.

[Chemical modification of phenylalanyl-tRNA synthetase and ribosomes of Escherichia coli with derivatives of tRNA-Phe carrying photoreactive groups on guanosine residues]. / Modifikatsiia fenilalanil-tRNKrhe, nesushchimi reaktsionnosposobnye gruppy na ostatkakh guanozina.

Photoreactive derivatives of tRNAPhe (E. coli) were synthesized by alkylation of the tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzylamine and subsequent treatment with 1.4--dinitro-5-fluorophenylazide. The derivatives are active in binding with ribosomes and phenylalanyl-tRNA synthetase when the extent of modification is lower than 3 reactive groups per tRNAPhe molecule. Under irradiation the derivatives modify exclusively the beta-subunit of the phenylalanyl-tRNA synthetase.

[Alkylation of tRNA-Phe free and bound to phenylalanyl-tRNA synthetase with 4-(N-2-chloroethyl-N-methylamino)benzylamine]. / Alkilirovanie tRNK-Phe s fenilalanil-tRNK-sintetazoi Escherichia coli s pomoshch'iu 4-(N-2-khlopetil-N-metilamino)-benzilamina.

Reactivities toward the alkylating reagent 4-(N-2-chloroethyl-N-methylamino)benzylamine of guanosine residues in the tRNAPhe free and bound to the phenylalanyl-tRNA synthetase were determined. This highly efficient reagent modifies mainly guanosine in single-stranded as well as in helical regions of tRNAs. The reaction is sensitive to the elements of the macrostructure of tRNAs. It is found the phenylalanyl-tRNA synthetase protects the D-stem of the tRNAPhe (guanosines G10, G22, G24) from alkylation.

[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.

[Selective binding of tRNA by RNA dependent DNA-polymerase from Escherichia coli]. / Izbiratel'noe sviazyvanie tRNK RNK-3avisimoi DNK-polimerazoi Escherichia coli.

Highly purified RNA dependent DNA-polymerase was isolated recently from E. coli by Romashchenko et al. [8]. The present data demonstrate that total E. coli tRNA inhibits poly(dT) synthesis on poly (A): oligo (dT) catalyzed by the enzyme when the enzyme:tRNA ratio is about 1 : 80--100. The inhibition results from the binding of certain tRNA's by the enzyme. The enzyme tRNA complex was separated from the unbound tRNA's by gel-filtration of Sephadex G-100. The tRNA's extracted from the complex are able to inhibit completely poly(A):oligo(dT) templated synthesis of poly(dT) under the enzyme:tRNA ratio about 1 : 2--3. Aminoacylation of tRNA separated from the enzyme complex has shown that E. coli RNA dependent DNA-polymerase selectively binds tRNAThr and to a lesser extent tRNATyr and tRNALys. It is suggested that the enzyme bound tRNA's carry out the functions of natural primers which compete with oligo(dT) for the enzyme responsible for the primer binding.

[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.

[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 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.

[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.

[Substrate specificity of restriction endonuclease VspI]. / Opredelenie substratnoi spetsifichnosti restriktsionnoi éndonukleazy VspI.

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.

[The nucleotide sequence of the 3'-terminal region of encephalomyocarditis virus RNA]. / Posledovatel'nost' nukleotidov 3'-kontsa RNK virusa éntsefalomiokardita.

Reverse transcription afforded DNA-copies of the 3'-terminal sequence of mouse encephalomyocarditis virus RNA. Cloning of this cDNA in pBR 322 plasmid and sequencing of DNA of one of the clones established the structure (552 nucleotide residues) of the 3'-terminus of viral RNA. This region contains a non-translatable fragment (126 nucleotides) and also the fragment coding for the C-terminus of viral replicase.

[Nucleotide sequence of the 3'-terminus of encephalomyocarditis virus RNA]. / Nukleotidnaia posledovatel'nost' 3'-kontsevoi oblasti RNK virusa entsefalomiokardita.

Reverse transcription produced DNA-copies of the 3'-terminal sequence of mouse encephalomyocarditis virus RNA. Cloning and sequencing of this cDNA afforded the structure of the 3'-terminus of viral RNA over the length of 2988 nucleotide residues. The probable sites of proteolytic cleavage of the immature protein have been determined and the homology with the respective poliovirus proteins has been established.

[Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends]. / Plazmidy pMB123 i pMB124--vektory dlia polucheniia subfragmentov DNK s prizvol'nymi "lipkimi" kontsami.

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.

[Primary structure of fragment 7 of the influenza virus A/USSR/90/77(H1N1) RNA]. / Pervichnaia struktura segmenta 7 RNK virusa grippa A/USSR/90/77(H1N1).

The double-stranded DNA copy of the matrix protein (M) gene of the influenza virus A/USSR/90/77 (H1N1) has been inserted in PstI site of plasmid pBR322 and cloned in E. coli. The full-length DNA copy of the M gene has been sequenced using Maxam-Gilbert method. Analysis of nucleotide sequence of segment 7 RNA of influenza virus A/USSR/90/77 and nucleotide substitutions, as compared with known primary structures of segment 7 RNA for other strains, is presented. A hypothetical model of secondary structure of segment 7 RNA of influenza virus and repeating sequences at nucleotide and amino acid levels, revealed in the central region of M1 protein, are discussed.

[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.