Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses.

[Testing of apathogenic influenza virus H5N3 as a poultry live vaccine].

Four H5N2 experimental vaccine strains and the apathogenic wild duck H5N3 influenza virus A/duck/ Moscow/4182/2010 (dk/4182) were tested as a live poultry vaccine. Experimental strains had the hemagglutinin of the A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site or the hemagglutinin from attenuated virus (Ku/ at) that was derived from the highly pathogenic influenza virus A/chicken/Kurgan/3/2005 (H5N1). The hemagglutinin of the Ku-at has the amino acid substitutions Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2, while maintaining the polybasic HA cleavage site at an invariable level. The other genes of these experimental strains were from the H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). A single immunization of mice with all tested strains elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal dose of A/chicken/Kurgan/3/05. The pathogenicity indexes of the Ku-at and the other strains for chicken were virtually zero, whereas the index of the parent H5N1 virus A/chicken/Kurgan/3/2005 was 2.98. Intravenous, intranasal, and aerosol routes of vaccination were compared. It was shown that the strain dk/4182 was totally apathogenic for one-day-old chicken and provided complete protection against the highly pathogenic H5N1 virus.

Characteristics of pigeon paramyxovirus serotype-1 isolates (PPMV-1) from the Russian Federation from 2001 to 2009.

Monitoring programs for highly dangerous avian diseases in the Russian Federation from 2001 to 2009 detected 77 samples that were PCR positive for avian paramyxovirus serotype-1 (APMV-1) from sick or dead feral and domestic pigeons. Nucleotide sequences of the fusion (F) gene, including a nucleotide sequence encoding the F protein cleavage site, were determined for these isolates. All of the studied isolates possessed virulent F0 protein cleavage sites (112KRKKRF117, 112RRQKRF117, or 112KRQKRF117). Intracerebral pathogenicity index (ICPI) values determined for seven of the isolates exceeded the value of 0.7 (the range from 0.8 to 1.41). Based on partial genome sequencing and phylogenetic analysis, the isolates were assigned to two individual sublineages within class II genotype VIb. It was determined that most of these Newcastle disease virus isolates (70/77) recovered from the pigeons belonged to a relatively poorly studied sublineage VIb/2. The complete nucleotide sequence of the genome for the Pigeon/Russia/Vladimir/687/05 isolate of sublineage VIb/2 was determined.

[The generation and characteristics of reassortant influenza A virus with H5 hemagglutinin and other genes from the apathogenic virus H6N2].

The experimental reassortant vaccine strain VN-gull (H5N2) containing H5 hemagglutinin (HA) with a removed polybasic site in the connecting peptide and other genes from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (gull/M) was obtained using a two-step protocol. At Step 1, the reassortant with HA of A/Vietnam/1203/04-PR8/ CDC-RG and other genes from cold-adapted A/Leningrad/17/47 (VN-Len) viruses was generated due to selection with antibody to H2N2 at 26 degrees C. At Step 2, the reassortant VN-gull was obtained by replacing all genes from Len with those from gull/M due to selection with antibody to H6N2 at 39 degrees C. The reassortant VN-Len was apathogenic and the reassortant VN-gull was weakly virulent in mice. Both gave rise to specific antibodies and 4 weeks after single inoculation they provided complete protection against further challenge with highly pathogenic HSN1 virus A/chicken/Kurgan/3/05 (H5N1) (Ku-Len). The chickens infected with live VN-gull virus showed neither clinical symptoms, nor fecal virus excretion; nevertheless, they gave rise to antibodies and were protected from the further challenge with A/chicken/Kurgan/3/2005. The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

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.

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.

[S3 gene fragment sequence analysis in the chicken reovirus isolates detected in the poultry farms of the Russian Federation].

The paper presents data on the comparative analysis of nucleotide sequences of a S3 gene fragment of 67 chicken reovirus (CRV) isolates from the abnormal biopsy specimens tested in 1999 to 2007. These CRV isolates were ascertained to differ from vaccine strains in the nucleotide sequence of the S3 gene. The approximate substitution rates for the S3 gene were established to range from 2.0 x 10(-3) to 6.0 x 10(-3) nucleotide substitutions per year.

[Epizooty caused by high-virulent influenza virus A/H5N1 of genotype 2.2 (Qinghai-Siberian) among wild and domestic birds on the paths of fall migrations to the north-western part of the Azov Sea basin (Krasnodar Territory)].

Isolation, followed by the sequencing the full-size genome of strains of A/chicken/Krasnodarl300/07 and A/Cygnus cygnus/Krasnodar/329/07, has shown that they belong to genotype 2.2 (Qinghai-Siberian). The strains were deposited at the State Virus Collection of the Russian Federation and nucleotide consequences were at the International databank GenBank. The strains contained 10 unique amino acid replacements in reference to the consensus of the Qinghai-Siberian genotype in the PB2, PA, HA, NA, and NS1, which suggests that regional variants may form in different parts of an area.

Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay.

The recombinant antigen obtained by cloning and expressing two IBV nucleocapsid protein fragments (143-414 aa, 281-414 aa) in Escherichia coli was used for the detection of avian infectious bronchitis virus (IBV) specific antibodies in chicken sera by the indirect ELISA (rNpIBV-ELISA). As a result of testing 1524 serum samples the diagnostic sensitivity and specificity of rNpIBV-ELISA when comparing those of the routine whole IBV ELISA have been shown to be 93.81% and 87.36%, respectively. The agreement value was 91.5%.

[Differentiation of strains and isolates of chicken reovirus using PCR and heteroduplex mobility assay in amplified cDNA].

The method of chicken reovirus strain differentiation was worked out on the basis of RT-PCR and heteroduplex mobility assay (HMA). The S3 gene cDNA (633-896 b.p.) of some Russian and Italian chicken reovirus isolates was amplified by RT-PCR. The analysis of these cDNA samples was carried out by HMA. The relation between nucleotide differences and relative mobility of compared cDNA heteroduplex was reflected by the regression curve. The equation of linear regression was derived (y = 91.726-0.89x; where y is the level of nucleotide difference of compared cDNA (%), x is the relative mobility of compared cDNA heteroduplex (%)). This method made it possible to take correct results within 5-35% of nucleotide difference in heteroduplex sequences.

[Biological properties of the chick infectious bronchitis virus isolated in Russia].

A field chick infectious bronchitis virus (IBV) was isolated from the pathological material on chick embryos. The nucleotide sequence of the S1 gene was determined and comparatively analyzed with some sequences of this gene of foreign and Russian vaccine strains and isolates. A cross-neutralization test using sera to various IBV seroptypes was performed. The isolate was shown to antigenically differ from the reference strains. Bioassay was carried out, by using one-day chicks and the immunogenic properties of the virus were investigated.

[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.

[Comparative analysis of the VP2 variable region of the gene from infectious bursal disease virus isolates]. / Sravnitel'nyi analiz variabel'noi oblasti gena VP2 izoliatov virusa infektsionnoi bursal'noi bolezni.

Variable cDNA regions in the VP2 gene of 24 isolates of infectious bursal disease virus (IBDV) isolated in Russia in 1993-1996 were amplified by the "nested" PCR and sequenced. The primary structure analysis of the VP2 gene variable region revealed 2 major groups of IBDV isolates. The first group consisted of the isolates with the structure identical or closely related to the highly virulent European strains CS89, 74/89A, 661, JY86, and DV86, the second group included the isolates with a high level of homology to the vaccine strains PBG98 and Cu-1. In addition, two isolates with original structure were identified, which differed from previously studied strains.

[Sequence analysis of hexon gene from adenovirus KR95 inducing hydropericardium syndrome in chickens]. / Analiz posledovatel'nosti gena geksona adenovirusa KR95, vyzyvaiushchego sindrom gidroksiperikardita u kur.

The nucleotide sequence of a part of the HindIII-D fragment (3300 b.p.) of adenovirus KR95 DNA has been determined. Analysis of the nucleotide sequence disclosed a continuous ORF for hexon gene (2814 b.p.) coding the 937 residue protein, part of ORF for the C-terminal region of pVI polypeptide, including 114 residues and the beginning of ORF coding 25 N-terminal residues for viral endoproteinase. Comparison of predicted KR95 hexon sequence and 8 mammalian and avian adenovirus hexon sequences revealed the highest homology between KR95 strain and avian adenoviruses FAV10 and FAV1 (91.1 and 80.1%, respectively). The results were used for creating a test system on the basis of the polymerase chain reaction. The system was used in analysis of fowl samples obtained from 12 poultry farms in Russia. The sequences of hexon gene amplified fragments in the isolated strains and similar fragments of other mammalian and avian adenoviruses have been compared.

[Primary structure of the F-gene from Rinderpest virus strain K]. / Pervichnaia struktura F-gena virusa chumy krupnogo rogatogo skota shtamma K.

Synthesis, cDNA cloning, and nucleotide sequencing of F gene of rinderpest virus strain K was carried out. Analysis of nucleotide sequence showed the only open reading frame coding for protein from 546 a.o. with mol. weight 58.6 kDa. The mean percentage of identical nucleotide residues between F genes of strains K, Kabete O, and L is 76.4% for 5'-untranslated region and 90.5% for translated region, the share of similar amino acid residues in the respective proteins is 92.9%. The structure of restriction site of F0 precursor protein in rinderpest strains with different virulence is similar. Protein F of rinderpest virus strain K has 3 potential glycosylation sites and 13 cystein residues in positions identical to those of F protein of rinderpest strains Kabete O and L.

[Cloning fragments of the RNA polymerase gene of an attenuated variant of the foot-and-mouth disease virus A22]. / Klonirovanie fragmentov gena RNK-polimerazy attenuirovannogo varianta virusa iashchura A22.

The cDNA fragments complementary to RNA-polymerase gene and 3'-untranslated genome region of attenuated foot-and-mouth disease virus strain A(22)645 have been synthesized and cloned into a plasmid vector pUC19 in E. coli JM109. The cloned cDNA fragments were characterized as to their size, orientation towards the plasmid, and localization in the virus genome. Restriction maps for complete gene and two cDNA clones were constructed.

[Primary structure of gene H of cattle plague virus strain K]. / Izuchenie pervichnoi struktury H-gena virusa chumy krupnogo rogatogo skota shtamma K.

Synthesis, cDNA cloning, and identification of H gene nucleotide sequence of rinderpest virus (RPV) K strain are carried out. Analysis of the identified nucleotide sequence has revealed the single open reading frame encoding a protein consisting of 609 amino acids with molecular weight of 68 kDa. The mean nucleotide homology between H genes of K, Kabete O and L strains in 88.0%, the mean amino acid homology of the corresponding proteins is 88.2%. RPV K strain hemagglutinin contains 5 potential glycosylation sites. The position of all 13 cystein bases is identical to positions in H proteins of RPV Kabete O and L strains. Studies of the hydrophobic profile of the compared proteins have shown 2 potential transmembrane fragments.

[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.

[The nucleotide sequences of the HN gene and F gene fragment of Newcastle disease virus strains BOR74 and BOR82]. / Nukleotidnye posledovatel'nosti gena HN i fragmenta gena F shtammov BOR74 i BOR82 virusa N'iukaslskoi bolezni.

The complete nucleotide sequence of HN gene, the region of F gene, and intergene regions (M-F, F-HN, and HN-L) of the BOR74 and BOR82 strains of Newcastle disease virus have been determined. Based on the nucleotide and amino acid sequences, the speeds of the nucleic and amino acid changes were calculated (approximately 10(-3) nucleotides or amino acids/year). The BOR strains were grouped phylogenetically with the asymptomatic strains. These strains and the BOR strains have the same motif of the cleavage site (112GKQGR116-L117), but the HN protein of BOR strains has the 572 amino acids which differ the BOR strains from all other strains (571, 577, and 616 amino acids).

[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.