Molecular and genetic characterization of avian laryngotracheitis virus isolates obtained in Ukraine

Aim. To conduct a virological, PCR, PCR-RFLP and sequencing study of infectious laryngotracheitis virus (ILTV)isolates obtained from sick and dead chickens at industrial and backyard poultry farms in the eastern region of Ukrainecollected over the years 2010–2019 and to establish their pathotype and relationship with internationally occurring strains.Methods. Material for virological studies was collected in the framework of research program of the NSC IEСVM during2010-2019 in the poultry farms in the North-Eastern region of Ukraine, where the birds with the respiratory clinicalsigns were found. In total, 28 poultry farms were observed. ILTV isolates were obtained with conventional methods,using 10–12-day-old chicken embryos. A 0,2 ml of 10–20 % suspension of pathological material in PBS was used forinoculation. For in-depth studies, we used 4 isolates of ILTV obtained from sick and dead chickens from industrialand backyard poultry farms in Kharkiv, Luhansk, Donetsk, and Sumy regions from 2010–2019. The identification ofILTV isolates was performed via conventional PCR. The pathotype of ILTV strains was determined using PCR-RFLP(polymerase chain reaction – restriction fragment length polymorphism) analysis. The PCR-RFLP was performed atRoyal GD, the Netherlands. The (partial) sequencing of the US8 gene was performed using Sanger sequencing method.The phylogenetic analysis, using sequences of 2 Ukrainian strains (MZ323228, MZ333273) and 17 international genesequences present in GenBank, was performed using the Maximum Likelihood method. For comparative analysis,sequences of vaccine ILT virus strains were used. Results. Over the years 2010-2019, 7 isolates of ILTV were obtainedfrom sick and dead poultry with typical clinical signs and internal lesions at industrial and backyard farms of the Kharkiv,Donetsk, Luhansk and Sumy regions, and the Autonomous Republic of Crimea. Other avian respiratory viral and bacterialpathogens were not detected. Five isolates were obtained from poultry of industrial holdings where vaccination againstILT is carried out. Using PCR-RFLP analysis of 4 isolates, we found that three of them (Sumy 6-11/19, A 04-12, B 2-10)to belong to vaccine-type ILTV strains and only one, B 59-11strain, belongs to wild-type ILTV. Vaccine-type ILTV strainscirculated and possibly still circulate in Ukraine in industrial and backyard poultry farms among both vaccinated and non-vaccinated poultry. An ILTV wild-type strain was obtained from non-vaccinated chickens from a backyard farm, whichmay indicate an important role of backyard farms in maintaining the circulation of the virus. After partial sequencing andphylogenetic analysis of the ILTV US8 gene the two Ukrainian strains studied were placed into two different clusters: Thevaccine-type B 2-10 strain, obtained from sick vaccinated chickens from an industrial farm, was close to vaccine-typestrains circulating in, China, Italy and the USA. The wild-type B 59-11strain, obtained from sick non-vaccinated backyardchickens, was located in another cluster and closest to a the wild-type B 59-11 ILTV strain from Brazil. Conclusions. Inthis article we describe for the first time the characterization of vaccine-type and wild-type isolates of ILTV in industrialand backyard poultry farms, proving their relevance for the poultry production in Ukraine. The results obtained show theneed and prospects for further monitoring of ILTV circulation in small backyard poultry farms and in industrial poultryfarms, especially following the frequent use for vaccination of live attenuated wild-type ILTV strains in Ukraine. Furthermolecular, phylogenetic and epidemiological characterization of the strains obtained should be performed in the nearfuture to further precise their attributes, epidemiology and origin.