Sequence analysis and development of attenuated vaccines from local infectious bronchitis virus strains
Date Issued
2007
Date
2007
Author(s)
Huang, Yuan-Pin
DOI
en-US
Abstract
Infectious bronchitis (IB) is an acute, highly contagious disease of chickens caused by infectious bronchitis virus (IBV). Since respiratory tract infections, including IB, are critical in the poultry industry, a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects avian viral respiratory pathogens in the same reaction was developed for the economic and clinical needs. Four sets of specific oligonucleotide primers for infectious laryngotracheitis virus (ILTV), IBV, Newcastle disease virus (NDV), and avian influenza virus (AIV) were used in this study. Expected products were generated from both multiplex RT-PCR and single RT-PCR. The detection limits of this multiplex RT-PCR were 3.5 ng, 0.14 ng, 0.4 ng, and 0.36 ng for ILTV, IBV, NDV, and AIV, respectively. Among the tested samples, seventy-seven percent of the 22 virus isolation-positive samples showed positive by genome detection, and ninety-one percent of the 64 virus isolation-negative samples showed negative by genome detection. This multiplex RT-PCR could be used for rapid screening and differential diagnosis of poultry viral respiratory infections. In addition to clinical diagnosis, this technique was also applied to the IBV detection in samples from two slaughter houses. Besides one vaccine isolate, twelve flocks showed positive IBV isolation from 82 chicken flocks (14.6%), including eleven TW I isolates and one novel genotypic isolate, but no TW II isolate. The estimated total chicken isolation rate was 1.6% to 3.1%. The high IBV isolation rate from healthy chickens indicates that sampling in the slaughter houses is a convenient and effective way for IBV monitoring.
To investigate the molecular epidemiology of IBV in Taiwan, an old IBV strain isolated in 1964 and other 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on the results of phylogenetic analysis, 13 strains were selected for sequencing the entire S1 and partial N genes. The results indicated that most Taiwan IBV strains could be divided into two distinct lineages, Taiwan Group I (TW I) and Taiwan Group II (TW II). Only one Massachusetts strain, 2994/02, and one China-related strain, 2992/02, were detected. No recombination was found between H120 and the Taiwan strains in the S1 gene, and neither was between Beaudette CK and Taiwan strains at the 3’ genome. However, the S1 gene showed a noticeable higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination of strain 2992/02 and 2994/02 has occurred.
Since most Taiwan isolates are unique, and vaccination of chicks with imported vaccines fails to protect chicks from IBV infections in Taiwan. In order to control IBV infection, selected Taiwan strains were attenuated through SPF chicken embryonated eggs. A TW I strain 2575/98 was passaged 74 times through SPF embryonated eggs, and then tested in SPF chickens. The attenuated vaccine was not pathogenic in 1-week-old chicks, had a neutralization index (NI) of greater than 4.4 and showed a protective rate of 90% when inoculated birds were challenged with a field IBV strain. Similar results were obtained for a vaccine made from a TW II IBV strain 2296/95. Additionally, the TW I attenuated vaccine strain had no reversion to virulence after five back passages in chicks. Therefore, these two IBV strains were transferred to a manufacturer for the vaccine production and evaluation. The safety and the efficacy of this attenuated strain were also tested in SPF chickens. The results of evaluation indicated that the attenuated strain 2575/98 at 102.3, 103.3, or 104.3 embryo infectious dose (EID50)/dose was not pathogenic to 3-to-10-day-old chicks. After vaccination in SPF chickens, the NI in vaccinated with 102.3, 103.3, or 104.3 EID50/dose were greater than 2, and in control groups were lower than 1. No bacteria, Newcastle disease virus, or avian influenza virus contamination was found in this attenuated vaccine preparation. Similar results were obtained for attenuated strain 2296/95. In field tests, the groups vaccinated with the attenuated strain 2575/98 or 2296/95 showed better performance in NI, livability and weight comparing to whom vaccinated with vaccine H120. In conclusion, the attenuated IBV 2575/98 and 2296/95 have potential for controlling IBV infection.
Furthermore, the sequences in the 3’ 7.3 kb of the genome amplified using RT-PCR before and after attenuation were compared to study the relationship between virulence and the sequences of three IBV strains, 1171/92, 2575/98 and 2296/95. After attenuation, two to six amino acid substitutions were found in the S1 subunit, and two or three amino acid substitutions were found in the S2 subunit. No or one amino acid substitution was found in the small membrane protein, and one or three amino acid substitutions were found in the membrane protein. However, no amino acid substitution was found in the N protein, indicating that the N protein might not be related to this attenuation. The un-translated nucleotide sequence after the N of one strain was partially deleted after attenuation, and might be correlated with virulence. This study is the first demonstration comparing sequence changes in the 3’ 7.3 kb of the genome of IBV after attenuation. The above mentioned information might be useful in future virulence studies.
This study describes the IBV molecular epidemiology, and develops two vaccine strains for controlling IBV infection in Taiwan.
Subjects
減毒疫苗
基因分析
傳染性支氣管炎病毒
多引子反轉錄聚合酶
鏈鎖反應
屠宰場
Attenuated vaccine
Gene analysis
Infectious bronchitis virus
Multiplex RT-PCR
Slaughter house
Taiwan Group I
Taiwan Group II
SDGs
Type
thesis