Genetic analysis of genotype G57 H9N2 avian influenza virus isolate A/chicken/Tajikistan/2379/2018 recovered in Central Asia.

This paper presents genetic data on the full genome analysis of A/chicken/Tajikistan/2379/2018 H9N2 influenza virus isolated in September 2018 from chicken pathological material received from poultry farms of the Republic of Tajikistan and subtyped as H9N2 by serological and molecular methods. According to the results of hemagglutinin gene sequencing, the amino acid sequence of the cleavage site was RSSR/GLF, which is typical for low-virulent avian influenza virus. Phylogenetic analysis of the nucleotide sequence of a hemagglutinin gene fragment (nt 1-1539 of the open reading frame) showed that the A/chicken/Tajikistan/2379/2018 H9N2 isolate belongs to the Y280 genetic group of low-virulent A/H9 influenza virus, which is widespread in Southeast Asia. The complete nucleotide sequence of the viral genome was determined. Comparative analysis of all genomic segments revealed that the A/chicken/Tajikistan/2379/2018 H9N2 virus is closely related to an A/H9 influenza virus isolated in the Far East of the Russian Federation in 2018. Genetic similarity (97.1-99% identity in four out of eight viral genes) was found to isolates of an H7N9 subtype virus recovered in the Inner Mongolia and Hebei regions of China in 2017. According to the analysis of the predicted amino acid sequence of the studied isolate, the positions of some molecular markers indicate possible adaptation of the virus to mammals. Further genetic analysis showed that this virus belongs to genotype G57.

Armoured exogenous internal control for real-time PCR diagnosis of avian influenza.

An exogenous "armoured" PCR internal control (IC) short RNA was analyzed in conjunction with real-time RT-PCR method for diagnosis of avian influenza. The resistance to nucleases and increased physical stability of the IC was ensured using branched polyethyleneimine (PEI) which was in complex with IC-RNA. The option to add the IC directly to pathological material suspensions allows measurement of the nucleic acids extraction efficiency. Stability of armoured RNA-IC during storage and tissue suspension preparation was shown. The advantage of exogenous "armoured" IC was demonstrated in the experiment with AIV genome detection by qPCR in samples from different species of wild birds. The exogenous IC gave reproducible homogeneous Ct values in all tests.

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.