The Pathogenicity Evaluation Of Infectious Larynotracheitis Virus And The Genome Sequence Analysis Of Two Isolated Strains

A respiratory disease which caused by infectious laryngotracheitis virus has the feature of high contacting and acute infection. The disease is called Infectious laryngotracheitis. The chicken which are actue infection have the symptom of high morbidity and high mortality, The chicken which are chronic infection have the symptom of conjunctivitis and decreased of egg production and.egg quality. Since the disease is first reported in 1925, it is epidemic in the worldwide, nowadays, it is popular in many regions in our country.In order to clarified the change of the pathogenicity of ILTV LJS09 strain chicken embryo source virus, which has adapted to the LMH cells, we infected the SPF chickens with chicken embryo source virus and virus adapted to LMH cells four generations. Through evaluation the index of clinical symptom, clinical score, clinical index(1.4 and 0.7), morbidity(80% and 60%)and mortality(10% and 0), pathological anatomical change and the histopathologic morphology of chicken which infected with chicken embryo source virus, the change of serum antibody levels, the tropism organs of ILTV and the distribution of ILTV in the internal organs of infected chickens, and then evaluation and comparison the pathogenicity of the two. The result illustrate that those two infected groups have similar clinical symptom, pathological anatomical, the change trend of serum antibody level s and the tropism organs of ILTV. But the clinical score, clinical index, morbidity and mortality, the serum antibody level of chicken which infected with chicken embryo source virus is higher than chicken which infected with LMH cells adapted virus. This indicated that the pathogenicity of ILTV LJS09 strain chicken embryo source virus is higher than the LMH cells adapted virus. The study will provide some information to understand the pathogenicity of Chinese ILTV strains.We isolated two ILTV field strains in 2014 from Heilongjiang and Hebei provinces, named LHLJ1403 and LHB1409 strains, respectively. After separation from chicken embryo, identification the field strains by PCR and sequenced, and make those two field strains adapted to the LMH cells, then the purified virus was obtained through plaque purification on LMH cells. In order to investigate the Epidemiological trend of Chinese ILTV epidemic strains, we determined and analyzed the complete genome sequences of those strains LHLJ1403 and LHB1409 by PCR. The complete genome sequences are fragmented and formed 106 short fragments. We used 106 pairs of primers to amplify the complete genome sequences of LHLJ1403 and LHB1409.because the feature of IRS and TRS is inverting and repeating, The location and sequences of the repeat regions were determined using anchored PCR and nested PCR.The complete genome sequences were obtained through splicing all the fragments. The LHLJ1403 strain is 153222 bp long and has 79 ORFs, the LHB1409 is 153213 bp long and also has 79 ORFs. The DNAStar、MEGA、DNAMAN and ORF Finder biology microsofts were used to align the strains.(the reference strains were LJS09, SA2, A20, LTBlen, 1874C5, USDA, 81658, 63140, Laryngo, Serva). Through the homologous comparison between the 12 strains, the homologous between LHLJ1403 and LHB1409 strains were 98.82%, LHLJ1403 and LT Blen strains have the highest similarity up to 99.49%, LHB1409 and 63140 strains have the highest similarity up to 99.19%. Compared with SA2 and A20 strains, there were four fragments deletion within the unique long region(UL) of LHLJ1403 and LHB1409 strains, with the deletion of 14 bp in 693-706, 189 bp in 3297-3485, 19 bp in 3672-3690 and 32 bp in 4662-4693. There is a large fragment deletion 857 bp in the repeat regions of LHLJ1403 and LHB1409 strains compared with USDA strain, and compared with Serva strain, there also was a fragment deletion 217 bp in the repeat regions of LHLJ1403 and LHB1409 strains. what’s more,there are origin binding protein(OBP) exist and no AT-rich region in DNA replication region of ILTV LHLJ1403 and LHB1409 strains. Compared with the USAD strain, there are amino acid mutations in the glycoprotein,g C, g B, g D and g H/g L which can influence the cells tropism in the strains of LHLJ1403 and LHB1409. Compared the complete genome sequence of reference strains with the isolated strains, and drawn the Phylogenetic tree, we can found that LHLJ1403 has closer relationship with USA vaccine strains LT Blen and Laryngo and they are in the same branch, LHB1409 has closer evolutionary relationship with vaccine strains Serva and USA virulent strains 63140 and they are in the same branch.In this research, the complete genome sequences of those two Chinese isolated field strains have been sequenced and determined. Comparing and analyzing the sequence of those two strains with the virulent, attenuated and vaccine stains. These resulted provide a foundation for the research of epidemiological, genetic and molecular biology features, and provide some information to understand the epidemiological of ILT, and develop the new generation vaccines to manage the disease.