Biological Characteristics of Infectious Laryngotracheitis Viruses Isolated in China

Abstract

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens and results in huge economic losses in the poultry industry worldwide. To correlate the genomic difference with the replication and pathogenicity, phenotypes of three ILTVs isolated from chickens in China from 2016 to 2018 were sequenced by high-throughput sequencing. Based on the entire genome, the isolates GD2018 and SH2017 shared 99.9% nucleotide homology, while the isolate SH2016 shared 99.7% nucleotide homology with GD2018 and SH2017, respectively. Each virus genome contained 82 ORFs encoding 77 kinds of protein, 31 of which share the same amino acid sequence in the three viruses. GD2018 and SH2017 shared 57 proteins with the same amino acid sequence, while SH2016 shared 42 and 41 proteins with the amino acid sequences of GD2018 and SH2017, respectively. SH2016 propagated efficiently in allantoic fluid and on chorioallantoic membranes (CAMs) of SPF chicken embryo eggs, while GD2018 and SH2017 proliferated well only on CAMs. GD2018 propagated most efficiently on CAMs and LMH cells among three isolates. SH2016 caused serious clinical symptoms, while GD2018 and SH2017 caused mild and moderate clinical symptoms in chickens, although the sero of the chickens infected with those three isolates were all positive for anti-ILTV antibody at 14 and 21 days after challenge. Three ILTVs with high genetic homology showed significant differences in the replication in different culture systems and the pathogenicity of chickens, providing basic materials for studying the key determinants of pathogenicity of ILTV.

Keywords: biological characteristics; genome; infectious laryngotracheitis virus; pathogenicity; replication.

Figure A1

Diagram of proteins of three ILTVs. Each circle stands for 77 proteins of one ILTV, and the overlap part stands for the proteins with the same amino acid sequences of two or three ILTVs.

Figure A2

Phylogenetic tree of ILTVs. Phylogenetic analyses were performed using the MEGA6.06, based on the entire genome sequences of ILTVs. The strain of V1-99 was placed as the root branch in the phylogenetic tree. The scale bar indicates the number of substitutions per site. The “solid dot” indicates the ILTV isolates studied in this study. The “solid triangle” indicates the other ILTV strains isolated in China.

Figure A3

Proliferation of ILTVs in different cultural systems. A. The virus titers in the supernatant of LMH cell culture in T25 flasks inoculated with ILTVs at MOI of 0.01 were tested at different time points. B. The virus titers in the supernatants of homogenized CAMs were tested at different time points after SPF chicken embryonated eggs were inoculated with 100 EID₅₀ of ILTVs on the CAMs through artificial air sacs. C. The virus titers in the allantoic fluids were tested at different time points after SPF chicken embryonated eggs were inoculated with 100 EID₅₀ of ILTVs into the allantoic cavity. Asterisks indicate a statistically significant difference between the titers of the virus. p-values were calculated based on a two-tailed, unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure A4

Clinical index of chickens infected with different ILTV isolates. The clinical symptoms of individual chickens were evaluated on a scale of 0–4 according to the severity of the disease. The clinical index was calculated as the total score divided by the number of chickens.

Figure A5

The pathological changes in the larynx and tracheal of infected chickens. Three chickens in each group were euthanized, and their organs were observed by the gross anatomy method 6 days post-challenge. Three SH2016-infected and one of the SH2017-infected chickens showed clear hemorrhagic tracheitis with blood clots. The laryngotracheal lesions of the other 2 chickens infected with SH2017 and the 3 infected with GD2018 were unclear.

Figure A6

The antibody induced in chickens by ILTVs. To detect the immune response of chickens infected with different ILTV, the sero were collected at 0, 14, and 21 days post-challenge to measure antibodies by ELISA assay. The samples with the OD₄₅₀ values less than 0.3 were considered negative for anti-ILTV antibodies. All of the sero of chickens infected with ILTVs converted to be positive to anti-ILTV antibodies at 14 and 21 days post-challenge.