Adaptation of infectious bronchitis virus in primary cells of the chick embryo chorioallantoic membrane

The susceptibility of the primary chick embryo chorioallontoic membrane cells to infectious bronchitis virus was evaluated after twenty consecutive passages in chick embryo chorioallontoic membrane cells. Virus replication was monitored by cytopathic observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction (RT-PCR). At 72 hours post-infection (p.i.) in third passage, the cytopathic effect was characterized by rounding up of cells, monolayer detachment, intracytoplasmic brownish colouration was readily observed by immunoperoxidase from 24 hours p.i in third passage, and at all times the extracted viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Tissue culture ineffective dose50 (TCID50) was used to measure virus titration performed on primary chick embryo chorioallontoic membrane cells and the titre in twenty passage was 10 TCID50/ml. The results obtained in this study suggested that the primary chick embryo chorioallontoic membrane cells can be used for adaptation infectious bronchitis virus (IBV) and may be considered a step forward for the use of these cells in the future for IBV vaccine production


Introduction
Infectious bronchitis virus (IBV) infects the respiratory tract, kidneys and oviduct of chickens of all ages, causing retarded growth, mortality, reduced egg production and inferior egg shell quality and in many countries the disease remains one of the main problems affecting existing or developing poultry industries.Until now, there is no cure for the disease (1).Prevention is to import birds from disease-free flocks only or through vaccination, broilers are normally vaccinated at 1 day of age with live attenuated vaccines (2).In addition, breeders and egg layers are also vaccinated at approximately 8 week intervals with live attenuated vaccines, and with inactivated vaccines after they start laying eggs (3).Acute Infectious bronchitis virus are usually detected by the indirect immunoperoxidase test, enzyme-linked immunosorbent assay, virus isolation or serological approaches (4,5).However Infectious bronchitis virus infections can also be diagnosed by detection of viral RNA by RT-PCR, which make the diagnostic rapid and also dependable (6,7).Usually, Infectious bronchitis vaccines have been produced by growing vaccine virus strains in embryonated chicken eggs.Infectious bronchitis virus is harvested from the allantoic fluid and used to create a vaccine, nevertheless this method has the disadvantages of being labour-dense, takes long time and requires large area for the incubation of eggs.Cell cultures on the other hand considered are more suitable and less expensive than eggs and also convenient to inspect microscopically for indication of viral proliferation (5,8).There is therefore an urgent need to improve on the current Infectious bronchitis virus vaccines production technologies based chick embryo chorioallontoic membrane (CAM) cells.The development of cell-culture platforms as an alternative to the eggs for the manufacture of IBV vaccines is likely the most rapid and promising solution to overcome current vaccines production.In this study, firstly we investigated here for the first time the ability of IBV to grow in chorioallontoic membrane cells and second to detect titres of IBV in chorioallontoic membrane cells plus the possibility to use these cells for vaccine production in the future

Virus inoculation
The stock of IBV was originally obtained from the Faculty of Veterinary Medicine, University Putra Malaysia.This virus was isolated from the allantoic fluid of embryonated chicken eggs.Initially, 0.3 ml of the virus stock was diluted in 30 ml of 10x PBS giving a dilution of 1:100.This diluted virus was used to inoculate primary chick embryo chorioallontoic membrane cells.Fully confluent 25 cm 2 flasks of cells were used for virus passage in an attempt to adapt the virus to replicate in these cells.

Cells and media
Primary chick embryo chorioallontoic membrane (CAM) cells were prepared from 9-to 10-day-old specific pathogenic free (SPF) chick embryo.The chorioallontoic membranes were collected, washed with phosphate buffer saline and digested with trypsin/EDTA.The reaction was stopped by adding (Dulbecco's Minimal109 Essential Medium (DMEM) complete growth medium (GIBCO Laboratories, USA) supplement with; 2.0 g NaHCo3, 10% fetal calf serum (FCS) and 1% antibiotic of penicillinstreptomycin.After centrifugation at 1000 g for 10 min, the chorioallontoic membrane cells were resuspended in the same medium and filtered through sterile gauze.The chorioallontoic membrane cells in the filtrate were distributed on plastic tissue culture flasks and incubated at 37 °C with 5 % CO2.

Tissue culture infective dose 50 (TCID 50 )
The infectivity of replicate infectious bronchitis virus to chorioallontoic membrane cells were determined by calculating 50% end point, as described by Reed and Muench (9).Ten-fold serial dilution of IBV was prepared in phosphate buffer saline phosphate buffer saline from 10 -1 to 10 -10 .A 96 well tissue culture microtiteration plate (Titertek, UK) was used to prepare chorioallontoic membrane cells monolayers.A 100 μl of each virus dilution was added in each well of first row leaving last two wells as negative control.The plate was incubated at 37°C for 1 hour to allow adsorption.Then 100 μl of prewarmed maintenance medium was added in each well and again incubated at 37°C in 5% CO 2 .The plate was observed twice daily for CPEs.The cells were stained with 1% crystal violet solution to determine the CPE.The highest dilution of virus showing 50% CPEs was considered as end point to calculate TCID 50/ml .

Indirect Immunoperoxidase Staining Test
The indirect immunoperoxidase test (IIP) was done according to the method of de Wit et al. (4).The infected chorioallontoic membrane cells were fixed with cold methanol: acetone (50:50 volum/volum) for 5 mins.The glass slides were then immersed in 1% hydrogen peroxide in absolute methanol for 30 mins.The phosphate puffer salain was then added to the glass slide for 15 mins.The glass slides were then air dried.The hyper immune serum was diluted 1:1000 with PBS and added to the glass slide incubated for 1 hour in room temperature.The glass slides were then washed 3 times with phosphate puffer salain for 5 mins each.The rabbit anti-chicken IgG-HRP conjugated secondary antibody (Bio-Rad, USA) was then added to the glass slides (1:1000) and incubated for 1 hour at room temperature.DAB substrate solution (DAB reagent set, Invitrogen, USA) was then added to the glass slides and incubated for 10 minutes in a dark room.The slides were mounted with buffer glycerol and examined under light microscope.

RNA isolation
RNA was isolated from infected cells using commercial RNeasy Mini Kit (Qiagen, USA) as recommended by the supplier.

Detection of PCR products
PCR products were separated in 1.5% agarose gel in 1 x TAE buffer stained with ethidium bromide, compared with molecular mass marker and visualized by ultraviolet (UV) transillumination.

Cytpopathic effect (CPE)
Infected cell monolayer steadily became broken as the virus proliferates to occupy extra cells in culture.In the first and second passage, the infectivity was sluggish and not very clear as the virus was just begin to adapt on cells.Through the third passage, we were able to visualize cytopathic effect but it was not whole (localized) the cells.During the fourth and fifth passage, cytopathic effect was rapid and detached in two days.It was characterised by rounding of cells, failure of cell adhesion, vacuolization in cells, clustering of infected cells (Figure 1).

Virus titration
The infectivity titre of the virus was found to increase gradually from the 5th to the 20 th passage.The TCID 50 titre was 8.6 after 20 passages (Table 1).

Indirect Immunoperoxidase Staining Test (IIPS)
In IIPS, brown complexes were seen as brownish intracytoplasmic granules around the nucleus (Figure 2) in infected chick chorioallontoic membrane cells cultures treated with IBV antiserum, but such effects was not seen in control cell cultures.

RT-PCR
RT-PCR of different passages at passages 2, 5, 10, 15 and 20 were performed.The expected 1025 bp was obtained for each passage examined (Figure 3).

Discussion
Infectious bronchitis (IB) virus, early described in 1930 (10), continues to be a main cause of disease in chickens of all ages and types in all parts of the world (11,12).The disease is occurring in all countries with a concentrated poultry industry, with the occurrence of infection close to 100% in most locations (13).Most of the countries rearing poultry commercially rely on vaccination to control IB, but the vaccine is still a great challenge for these countries because of the wide variety serotypes for this virus.
A large majority of the vaccines available are chick embryo-adapted vaccines.Cell cultures provide a useful alternative system for the virus preparation and adaptation, and find a suitable cell culture for IBV (14).In the present study, attempts were made to adaptation IBV isolate by blind passage first in SPF embryos (15) and then adapted to the CAM cells until 20 th passages.The cytopathic effects (CPEs) appeared after 72 hours of infection in third passage and this observation was also noticed by ( 16), but slightly varied from findings of Mahgoub et al. (12) where the IBV was adapted to Vero cell line after the third passage.
The difference might be due to the cell culture passage level of the virus strains used or variation in sensitivity of cell culture to different strains.
The total infectious titer passage 20 was found to be 10 8.6 TCID 50 /mL.These finding supports the previous report by (17) as they found titer 10 9 TCID 50 /mL in passage 25 in Vero cells.
The classical method of detecting adaptation and replication of IB virus in the cell culture using the cytopathic effect was augmented in this study with indirect immunoperoxidase is a relatively inexpensive and rapid method (18).Monoclonal antibody (Mab) reacting only with one or small number of epitopes of the IBV antigen, results in viral antigen was observed as brownish intracytoplasmic granules in CAM cells (18).
RT-PCR has been used widely as a rapid, sensitive, specific, and high throughput methodology for fast detection of genetic materials.This method was used successfully for detection of IBV (6,7).In this study RT-PCR performed for viral RNA extracted from infected CAM cells to amplify of 1025 bp.The use of this technique for virus detection has been reported previously (4).
The conclusion of this study that CAM cells have used for the first time to isolate and adapt IBV successfully which could open new horizons to use these cells for the vaccine production in the future.

Table1:Figure 1 :
Figure 1: (A) Uninfected control CAM cells monolayer.(B) Cytopathic effect of IBV isolate of the 3 rd passage at day 5 pi.The arrows show detachment of cells from the substrate with the eventual destruction of the entire monolayer.10 x.Bar = 200 µm.

Figure 2 :
Figure 2: Identification of IBV in CAM cells culture using infected cell cultures stained with HRP-conjugated antibody.(A) Uninfected control CAM cells.(B) CAM cells infected with IBV at 3 rd passage at day 5 pi.The arrow shows the presence of specific intracytoplasmic brownish coloration IBV antigens.10x.Bar = 100 µm.