We have previously shown that substitution of the spike (S) gene of the apathogenic IBV strain Beau-R by that of the pathogenic strain of the same serotype, M41, resulted in an apathogenic virus, BeauR-M41(S), which conferred protection against challenge with M41. We have constructed a recombinant IBV, BeauR-4/91(S), with the genetic base of Beau-R but expressing the spike protein of the pathogenic strain IBV 4/91(UK), which belongs to a different serogroup such as Beaudette or M41.
Similar to our previous findings with BeauR-M41(S), observations of clinical signs showed that the S gene of Avian infectious bronchitis virus Recombinant 4/91 did not confer pathogenicity to rIBV BeauR-4/91(S). Furthermore, protection studies showed that there was homologous protection; BeauR-4/91(S) conferred protection against wild-type 4/91 virus challenge as evidenced by the absence of clinical signs, IBV RNA assessed by qRT-PCR, and the fact that no virus was isolated. from tracheas removed from birds primarily infected with BeauR-4/91(S) and challenged with IBV 4/91(UK).
A degree of heterologous protection against the M41 challenge was observed, albeit at a lower level. Our results confirm and extend our previous findings and conclusions that protein S ectodomain swapping is a precise and efficient way to generate genetically defined candidate IBV vaccines.
Statement of Ethics
All animal testing protocols were carried out in strict accordance with UK Home Office guidelines and the license granted for experiments involving regulated procedures on animals protected by the UK Animals (Scientific Procedures) Act. of 1986. The experiments were carried out at the IAH Ministry of the Interior under license (PCD30/4301) and were approved by the IAH ethical review committee under the terms of reference HO-ERP-01-1, using chickens obtained from the IAH Poultry Production Unit.
Cells and viruses
The pathogenic strain of IBV 4/91 (UK) used in this study was a gift from Intervet UK Ltd and was cultured in 10-day-old specific-pathogen-free (SPF) Rhode Island Red (RIR) embryonated hen eggs obtained from the Institutes. poultry production unit; Primary chicken kidney (CK) cells are refractory to the growth of IBV 4/91 (UK).
M41-CK was derived from the pathogenic IBV strain M41 after adaptation in CK cells. Vaccinia viruses (VV) were routinely grown and titrated in Vero cells as described previously, while large stocks for DNA isolation were prepared from infected BHK-21 cells. Tracheal organ cultures (TOC) were prepared from 19-day SPF RIR chick embryos. Virus infectivity titers were performed in TOC and titers were expressed as 50% (median) cytostatic dose (CD50).
Recovery of an infectious EBV expressing a chimeric S protein
CVV-BeauR-4/91(S) DNA was purified and initially used to rescue rIBV in CK cells. Cell lysate (0.1 ml) from the infected and transfected CK (P0) cells were used to infect 10 day SPF embryos. Infected embryos were incubated at 37°C for 48 h, after which they were placed at 4°C overnight. Allantoic fluid (EP1) was collected and passaged a further five times on 10 days old SPF embryos and the resulting rIBV, BeauR-4/91(S), was used in subsequent experiments.
RNA was extracted from the allantoic fluid of infected eggs using the RNeasy® method (Qiagen) for amplification of part of the S gene by RT-PCR (Ready-To-GoTM RT-PCR beads) to confirm the identity of EBV by sequence analysis. A stock of BeauR-4/91(S) was produced in 10 days old SPF embryonated eggs, final titer 2×105.6 CD50 per ml, which was used for subsequent in vivo experiments.
In vivo analysis of rIBVs
Virus stocks for in vivo experiments were prepared from 10 days old SPF embryonated RIR eggs and titrated by TOC; stock virus titers were 4/91(UK) 5.4 log10 CD50, BeauR-4/91(S) 5.6 log10 CD50, and M41-CK 6.0 log10 CD50 in a volume of 1 ml. Five groups (n = 13) of 8-day-old SPF RIR chickens were used for in vivo analysis of EBV BeauR-4/91(S). Chickens were housed in negative pressure, temperature-controlled, HEPA-filtered isolation rooms, with each group housed in a separate room.
Three groups of birds were inoculated conjunctival (eye drops) and intranasally with 3.6 log10 CD50 of BeauR-4/91(S) in 0.1 ml of serum-free BES (N, N-Bis(2-hydroxyethyl) -2- medium containing aminoethanesulfonic acid). The other two groups were inoculated with BES medium without serum as controls. Three weeks after infection, the three groups that had been infected with BeauR-4/91(S) were challenged using 3.6 log10 CD50 in a total of 0.1 ml with IBV 4/91(UK), IBV M41 -CK or mock-challenged and the two mock-infection groups were sham-challenged or 4/91 (UK); in all cases, challenge viruses were administered conjunctively and intranasally.
The clinical signs used to determine pathogenicity were clicking (sneeze-like sound), tracheal rales (sound emanating from the bronchi, also detected by vibrations when holding a chick), wheezing (dyspnea), runny nose, watery eyes and ciliary tracheal activity. Chicks were observed daily for clinical signs; the snicks were counted independently by two people during a period of 2 min.
Birds were checked individually for the presence of tracheal rales, nasal discharge, watery eyes, and wheezing. Tracheas were removed from three randomly selected chickens from each group at 4, 5, and 6 days post-challenge to assess ciliary activity. Ten 1 mm sections were cut from three different regions of each trachea and the level of cryostasis was determined from each tracheal section using light microscopy.
Tracheal sections stored in PBS were frozen, thawed, and homogenized using the Tissuelyser II (Qiagen). The resulting tracheal suspensions were centrifuged and the supernatants were used to infect TOC. Separate tracheal suspensions were prepared from three birds (except for mock-infected group: 4/91-challenged, n = 2) per sampling day (days 4, 5 and 6 post-challenge) for BeauR-4/91(S ):4/91 and BeauR-4/91(S): M41 groups.
Six TOCs were infected with 100 µl of the corresponding tracheal suspension. After infection at 37°C for 1 h, 0.5 ml of medium was added and the TOCs were incubated at 37°C for 7 days, during which they were regularly observed for ciliary activity. To compare ciliary activity results, ANOVA analysis followed by Dunnett’s posthoc multiple comparison test was performed using GraphPad Prism version 5.03.