Molecular and genetic characterization of avian laryngotracheitis virus isolates obtained in Ukraine
Authors: Veretsun A. , Stegniy B. , Rula O. , Bolotin V. , Stegniy A. , Gerilovych A. , Muzyka D. 
Published: June 2021

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 Ukraine
collected 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 during
2010-2019 in the poultry farms in the North-Eastern region of Ukraine, where the birds with the respiratory clinical
signs 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 for
inoculation. For in-depth studies, we used 4 isolates of ILTV obtained from sick and dead chickens from industrial
and backyard poultry farms in Kharkiv, Luhansk, Donetsk, and Sumy regions from 2010–2019. The identification of
ILTV 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 at
Royal 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 gene
sequences 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 obtained
from 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 bacterial
pathogens were not detected. Five isolates were obtained from poultry of industrial holdings where vaccination against
ILT 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 strains
circulated 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, which
may indicate an important role of backyard farms in maintaining the circulation of the virus. After partial sequencing and
phylogenetic analysis of the ILTV US8 gene the two Ukrainian strains studied were placed into two different clusters: The
vaccine-type B 2-10 strain, obtained from sick vaccinated chickens from an industrial farm, was close to vaccine-type
strains circulating in, China, Italy and the USA. The wild-type B 59-11strain, obtained from sick non-vaccinated backyard
chickens, was located in another cluster and closest to a the wild-type B 59-11 ILTV strain from Brazil. Conclusions. In
this article we describe for the first time the characterization of vaccine-type and wild-type isolates of ILTV in industrial
and backyard poultry farms, proving their relevance for the poultry production in Ukraine. The results obtained show the
need and prospects for further monitoring of ILTV circulation in small backyard poultry farms and in industrial poultry
farms, especially following the frequent use for vaccination of live attenuated wild-type ILTV strains in Ukraine. Further
molecular, phylogenetic and epidemiological characterization of the strains obtained should be performed in the near
future to further precise their attributes, epidemiology and origin.