Molecular characterisation and phylogenetic analysis of Marek's disease virus in Turkish layer chickens.

1. There is no current data about the genotypes of Marek's disease virus (MDV) in Turkish poultry flocks; hence, this study was performed to analyse CVI988/Rispens, turkey herpesvirus (HVT) vaccine viruses and MDV field viruses as well as to perform phylogenetic analysis of MDV in Turkish layer chickens. 2. In 2017 and 2018, a total of 602 spleen samples from 49 layer flocks were collected from the Marmara, West Black Sea and Aegean regions. DNA was extracted from the spleen samples and the samples were analysed by real-time PCR probe assay to detect CVI988/Rispens and HVT vaccine viruses and MDV field strains. Samples found positive for MDV by real-time PCR were subjected to PCR using the Meq gene primers for phylogenetic analysis. 3. Amongst 49 flocks, virulent MDV was detected in nine flocks. CVI988/Rispens and HVT vaccine strains were detected in 47 flocks and HVT in all 49 flocks. Splenomegaly, hepatomegaly and tumours in the oviduct were observed in chickens of affected flocks. Virulent MDV was detected in 120 out of 602 spleen samples. Sequencing and phylogenetic analyses showed that MDVs detected in this study were closely related to MDV strains from Italy, Poland, Saudi Arabia, Iraq, India and China but showed diversity with MDV strains from Egypt and Hungary. Multiple sequence analysis of the Meq protein revealed several point mutations in deduced amino acid sequences. Interestingly, CVI988/Rispens vaccine virus from China (AF493555) showed mutations at position 66 (G66R) and 71 (S66A) along with two other vaccine strains from China (GU354326.1) and Russia (EU032468.1), in comparison with the other vaccine strain CVI988/Rispens (DQ534538). The molecular analyses of the Meq gene suggested that Turkish field strains of MDV are in the class of virulent or very virulent pathotypes. 4. The results have shown that MDV still affects poultry health, and the phylogenetic and amino acid variation data obtained will help in vaccination and control strategies.

Phylogeny and evolution of infectious bursal disease virus circulating in Turkish broiler flocks.

The emergence of new infectious bursal disease virus (IBDV) variants can threaten poultry health and production all over the world causing significant economic losses. Therefore, this study was performed to determine IBDV molecular epidemilogy, VP2 gene variation, and corresponding pathological lesions in IBDV infected chickens in Turkey. For this, 1855 bursa of Fabricius samples were collected from 371 vaccinated broiler flocks. Atrophia and haemorrhages were seen in the bursa Fabricius of very virulent IBDV (vvIBDV) infected chickens. Partial VP2 gene was sequenced and phylogenetic, recombination, and evolutionary analyses were performed. 1548 (83.5%) out of 1855 of bursa of Fabricius samples were IBDV positive and 1525 of those could be sequenced. The recombination analysis did not detect occurrence of any recombination event among the Turkish strains. Among 1525 sequenced samples, 1380 of them were found to be classical strains. Among 1380 classical strains, 1317 were similar to IBDV 2512, 11 to Faragher 52/70, 40 to 228 E, and 12 to Lukert strain. Out of 1525 reverse transcriptase ploymerase chain reaction positive samples, 144 of them were found to be similar to vvIBDV-VP2 gene reported to GenBank previously. The phylogenetic tree performed on a broad sequence dataset demonstrated grouping of vvIBDV Turkish strains in three different clusters, including sequences collected also from Iraq and Kuwait (Cluster 1), Indian (Cluster 2), and a distinct Turkish-only cluster (Cluster 3). The evolutionary rate estimation on branches/clades including Turkish strain mirrored the expected one for RNA viruses and no significant differences were found among different considered branches. In conclusion, results of this study indicate that vvIBDV strains similar to those circulating in various countries in the Middle East are present and undergoing evolution in chickens from Turkish broiler flocks. This point needs to be taken into account in planning adequate control strategies.

First Molecular Characterization of Avian Metapneumovirus (aMPV) in Turkish Broiler Flocks.

Viral respiratory diseases, including avian metapneumovirus (aMPV), have a significant economic impact on poultry industries. The frequency and genotype diversity of aMPV in Turkish broiler flocks is not known at present. The aim of this study was to report the first molecular identification and phylogeny of aMPV, which is circulating in Turkish broiler flocks. Trachea tissue samples and tracheal swabs were collected from 110 broiler flocks distributed in different geographical regions in Turkey between March 2017 and March 2018. Detection of aMPV was confirmed with the use of universal reverse transcriptase (RT) PCR, and eight (7.2%) broiler farms were positive for aMPV. Sequence analysis of the G gene revealed the exclusive presence of subtype B viruses. Three field isolates clustered closely with a 2002 Israel isolate, indicating a potential transmission route between these two countries and through the Middle East. The remaining five field isolates were closely related to a vaccine strain, even though broiler flocks in Turkey are not routinely vaccinated against aMPV. Therefore, we speculate these five isolates could have originated from nearby vaccinated turkey farms. Additionally, the presence of some nucleotide substitutions compared to the reference vaccine sequence suggests prolonged circulation and evolution of the original vaccine virus or a vaccine subpopulation was selected under field conditions. This evidence emphasizes the need for further detailed and more systemic approaches to evaluate aMPV spread and evolution in order to design effective control strategies.

Nota de investigación- Primera caracterización molecular de metapneumovirus aviar (aMPV) en parvadas de pollo de engorde en Turquía. Las enfermedades respiratorias virales, incluido el metapneumovirus aviar (aMPV), tienen un impacto económico significativo en la industrias avícola. La diversidad de la frecuencia y el genotipo de aMPV en las parvadas de pollos de engorde en Turquía no se conocen en la actualidad. El objetivo de este estudio fue reportar la primera identificación molecular y la filogenia de un metapneumovirus aviar, que circula en parvadas de pollos de engorde turcos. Se recolectaron muestras de tejido de tráquea e hisopos traqueales de 110 parvadas de pollos de engorde distribuidas en diferentes regiones geográficas de Turquía entre marzo del 2017 y marzo del 2018. La detección de metapneumovirus aviar se confirmó con el uso de un método de universal transcriptasa reversa y PCR. Ocho (7.2%) granjas de pollos de engorde fueron positivas para metapneumovirus aviar. El análisis de secuencia del gene G reveló la presencia exclusiva de virus de subtipo B. Tres virus de campo se agruparon estrechamente con un metapneumovirus de Israel del año 2002, lo que indica una posible ruta de transmisión entre estos los dos países y el Medio Oriente. Los cinco metapneumovirus de campo restantes estaban estrechamente relacionados con una cepa de vacuna, a pesar de que las parvadas de pollos de engorde en Turquía no se vacunan rutinariamente contra metapneumovirus aviar. Por lo tanto, se especula que estos cinco metapneumovirus podrían haberse originado en granjas cercanas con pavos vacunados. Además, la presencia de algunas sustituciones de nucleótidos en comparación con la secuencia de la vacuna de referencia sugiere una circulación prolongada y la evolución del virus de la vacuna original o una subpoblación de la vacuna se seleccionó en condiciones de campo. Esta evidencia enfatiza la necesidad de enfoques más detallados y más sistémicos para evaluar la propagación y evolución de metapneumovirus aviar a fin de diseñar estrategias de control efectivas. Abbreviations: aMPV = avian metapneumovirus; cDNA = complementary DNA; F = fusion; G = attachment; RT-PCR = reverse-transcriptase PCR.