Genetic diversity of Mycoplasma gallisepticum field isolates using partial sequencing of the pvpA gene fragment in Russia.

The genetic diversity of the pvpA gene of Mycoplasma gallisepticum (MG) samples originating from commercial chickens was investigated. In the present study, we evaluated the genetic variability of 26 field samples of MG detected in commercial chickens and turkeys from 18 regions of Russia and compared them to the reference strains of MG available in GenBank. Genetic variability was evaluated by partial nucleotide sequencing of the pvpA gene, which encodes a putative cytadhesin protein. Comparisons with MG strains and isolates from the United States, Australia, China, and Iran using sequence analysis of PCR products showed that Russian MG field samples clustered more closely to each other than to the international reference MG strains. The MG pvpA sequences were found to be highly variable with a discrimination index of 0.975 for Russian field samples. No apparent cluster was found using the criteria of year or location of detection. DNA sequence polymorphism and size variation in the pvpA gene were shown among the Russian MG field samples and could be used for MG typing. These findings might help better understand the relationship among MG isolates from Russia and other countries.

Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry.

In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

[Phylogenetic analysis of swine fever virus strains]. / Filogeneticheskii analiz shtammov virusa chumy plotoiadnykh.

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.

[Strain differentiation of the Newcastle disease virus by reverse transcriptase-polymerase chain reaction and sequencing population exchange]. / Shtammovaia differentsiatsiia virusa n'iukaslskoi bolezni s pomoshch'iu obratnoi transkriptsii-polimeraznoi tsepnoi reaktsii i sekvenirovaniia: smena populiatsii.

A system for detection and strain differentiation of Newcastle disease virus (NDV) by reverse transcription of polymerase chain reaction (RT-PCR) (isolation of RNA, choice of primers for nested PCR, and purification of PCR products) and sequencing is developed and optimized. A nucleotide sequence of gene F site, coding for the F2/F1 cleavage site of F0 fusion protein and including several hypervariable regions, is determined for 10 Russian strains and vaccine strains. The data indicate a replacement of NDV populations in Russia and a rapid evolution of the virus. The origin of pathogenic NDV strains which have been circulating up to the present time is still unknown.

[Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22]. / Izuchenie molekuliarnykh osnov izmeneniia nekotorykh biologicheskikh svoistv virusa iashchura podtipa A22.

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.

[Bacterial synthesis of immunogenic epitopes of foot-and-mouth disease virus fused either to human necrosis factor or to hepatitis B core antigen]. / Bakterial'nyi sintez immunogennykh épitopov virusa iashchura v sostave gibridov s faktorom nekroza opukholei i kor-antigenom virusa gepatita B.

Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.

[A simple method for RNA isolation and purification]. / Prostol Metod Vydeleniya i Ochistki RNK

RNAs from Escherichia coli cells, Syrian hamster kidney cells, foot-and-mouth disease virus, and Newcastle disease virus were isolated using glass fiber filters GF/F or GF/C. The RNA was reversibly adsorbed on the filters in the presence of 2 M guanidine thiocyanate and 50% ethanol (or isopropanol) and eluted with water. The fraction composition of the isolated RNA depended on the guanidine thiocyanate and alcohol concentrations in the adsorption and washing procedures. The RNA preparations obtained by this method can be used in reverse transcription and reverse transcription-polymerase chain reaction without additional purification.

[Use of aerosol A-300 amd GF/F (GF/C) filters for purifying fragments of DNA, plasmid DNA, and RNA]. / Ispol'zovanie aérosila A-300 i fil'trov GF/F (GF/C) dlia ochistki fragmentof DNK, DNK plazmid i RNK.

Aerosil A-300 and GF/F (GF/C) filters were used for purifying plasmid DNA and intact RNA as well as for the recovery of DNA fractionated on agarose gels. In the presence of guanidinium thiocyanate Aerosil A-300 and GF/F (GF/C) filters selectively bind nucleic acids, while proteins, agarose polysaccharides and salts are washed off. Nucleic acids desorbed from the Aerosil and filters are available immediately as substrates: DNA for restriction analysis, ligation and sequencing; RNA--for RT-PCR.

[Primary structure of the gene for protein VP1 from foot-and-mouth disease virus serotype O1 isolated in the USSR]. / Pervichnaia struktura gena belka VP1 virusov iashchura serotipa O1, izolirovannykh v SSSR.

The nucleotides sequence has been determined for the viral RNA and some of its cDNAs coding for the major antigenic protein of the epidemic stomatitis O1-194 and O1-1618 VP1 viruses. The expressed microheterogeneity has been registered for the population of the strain O1-194.

[Primary structure of the gene for VP1 protein of the foot-and-mouth disease virus of Asia 1 serotype]. / Pervichnaia struktura gena belka VP1 virusa iashchura serotipa Aziia 1.

The nucleotide sequence of the cDNA for the viral RNA region coding for the main antigenic protein of the epidemic stomatitis virus of Asia 1 serotype has been identified. The amino acid sequences in the regions of VP1 protein antigenic determinants of the serotype Asia 1 virus and other serotypes viruses have been compared.