Virus isolation and identification
During the period from June 2008 to November 2010, IBV surveys were performed in 96 suspected but vaccinated flocks from eastern, southern, southwestern and central China. Documented clinical signs of the birds included respiratory and typical nephropathogenic symptoms. Viruses in the homogenized tissue pool of kidney and trachea of the field isolates were propagated by inoculating via the allantoic cavity of 10 day old SPF embryonated eggs for more than three passages. The embryos dying within 24 hours of inoculation were screened to be nonspecific deaths. The isolates caused stunting, dwarfing, curling of the embryo or the presence of urates in the mesonephros after 5 days incubation were indicative of IB virus replication. The viruses were further confirmed by RT-PCR assays. The allantoic fluids containing IBV isolates after 72 h post inoculation were harvested and preserved in liquid nitrogen.
Total RNA extraction of the allantoic fluid was completed using RNAiso reagent (TaKaRa Biotechnology, Dalian, China) according to the manufacturers' instructions. Reverse transcription polymerase chain reaction (RT-PCR) was carried out by PrimeScriptTM One-Step RT-PCR Kit in 25 μl reaction volume containing 20 μl of RT-PCR PreMix (reaction buffer, dNTPs, 2 μl of enzyme mix), 2 μl of extracted viral RNA and the specific primers (National standard, GBT23197-2008), one primer pair targeting the M gene (Ms: 5'-CCTAAGAACGGTTGGAAT-3', Mx: 5'-TACTCTCTACAC ACACAC-3') and another pair for the 3' UTR of genome (3's: 5'-GGAAGATAGGCATGTAGCTT-3', 3'x: 5'-CTAACTCTATACTAGCCTAT-3').
After IBV confirmation, virus recovery was performed as previous described . Five 1-day-old SPF White Leghorn chickens were intranasally inoculated with each isolate, respectively. All of the chicks were examined and recorded daily for clinical signs for 20 days post inoculation, the dead birds were necrospied for lesions of respiratory tract or nephritis. Finally, all the survivors were sacrificed and necrospied. All of the animal experiments were conducted in accordance with the guidelines of Guangdong Province on the Review of Welfare and Ethics of Laboratory Animals, and under the protocol (SCAU-AEC-2010-1102) approved by the Animal Ethics Committee of South China Agricultural University.
Vaccination-challenge of immunity trials and clinical application
Based on clinical records and sequence blast from our previous report, YL6 strain (GenBank accession numbers: GU938393 for S1 gene) isolated from Yulin city of Guangxi province in June 2009 (with high morbidity of 100% and mortality of 79%) was selected for preparation of inactivated oil-emulsion vaccine. Ten-day-old SPF embryonated eggs were inoculated with strain YL6 through five passages to yield a titre of 106 to 107 median embryo infectious dose (EID50), which were titrated using the method described by Liu et al (2006a). The harvested viruses were inactivated with 2/1000 formaldehyde for 36 h at 37°C, followed by testing for absence of infectivity by embryo inoculation, adjuvanted with mineral oil in the ratio of 1 to 2 finally.
Eighty 1-day-old SPF White Leghorn chickens were housed in biosecurity isolators under quarantine conditions in Wen's Foodstuff Group Company, and randomly allocated into 4 groups with 20 birds each. The protection effect of live attenuated vaccine XZB (Baoite Biopharmaceutical Corp. Ltd., Qingdao, China; Commercial vaccine, consisted of W93 and H120 strain) and YL6 vaccine were compared through the vaccination-challenge trials. The birds were inoculated intranasally with routine XZB or vaccinated intramuscularly with inactivated vaccine YL6. The detailed vaccination procedures were showed in Table 2. Group 4 was reared as vaccine-negative control.
Blood samples were collected on day 5, 10, 17 and 24. Serum antibody titers of 10 randomly selected chickens from each group were diluted (1: 500) and evaluated with a commercial total antibody ELISA kit (IDEXX Corporation, Westbrook, Maine, USA) as described by Liu et al . Optical density (OD) of each well was read in an ELISA plate reader at 650 nm. Serum-to-positive ratios were calculated, using the formula: SP ratio = (OD sample - OD negative control)/(OD positive control - OD negative control). From these SP ratios, individual serum titres were calculated according to the manufacturer's instructions.
Challenge test was performed in biosafety-level (BSL)-3+ lab after the final antibody test to examine whether or not the novel strain vaccines can provide protective immunity against the relevant infectious viruses. All of the chickens at 24 days old were infected by intranasal instillation of 105 × EID50 of virulent nephropathogenic HY51 strain (GenBank accession numbers: GU938386 for S1 gene) isolated from Heyuan city of Guangdong province (with high morbidity of 100% and mortality of 76%) in May 2009, the strain was belonged to the IBVs of predominant genotype . The chicks were examined daily for signs of infection for 30 days after inoculation. The virus isolation was carried out through tracheal swabs after three weeks post challenge.
Sufficient inactivated oil-emulsion vaccine YL6 was produced as described previously, and applied in some chicken farms in southern China, including Guangdong, Guangxi, Guizhou, Fujian and Sichuan province. In the flocks with epidemic history, the vaccination procedures were as follows, broilers were inoculated intranasally with routine XZB at 5 days of age, and vaccinated intramuscularly with 0.2 ml of inactivated vaccine YL6 at 10 days of age; Broilers were vaccinated intramuscularly with 0.3 ml of YL6 again at 20 days of age. In the non-epidemic flocks, broilers were inoculated with routine XZB at 5 days of age, and immunized with 0.2 ml of vaccine YL6 at 10 days of age.