Avian infectious bronchitis (IB) is one of the important and economically-impactful diseases in the poultry industry that affects broiler chickens, pullets, layers, breeders.
The cause of IB is a Gamma coronavirus that has a single-stranded RNA genome with a length of ~27.6 kb [1] . The genome consists of 5-pol-S-E-M-N-3 components, the most important of which is the spike gene (S). This gene plays a major role in the production of neutralizing antibodies and determines the genotype of the virus based on the S gene sequencing. According to the high mutations and recombination events in the genome of the IBV, especially the S gene, new genotypes of the IBV are constantly emerging. One of these emerging genotypes is variant 2 (GI-23/IS-1494) which originally emerged in Israel, 1994, as a nephropathogenic genotype [2, 3] and is currently reported in different countries in the Middle East including Iran, Afghanistan, Iraq, Oman, Turkey, Jordan, and Europe [4] [5] [6] [7] [8] . Cross-protection studies have revealed that once a Massachusetts type vaccine is used, the protection level is about 25%. However, the combined usage of Mass type and 793/B vaccines in the vaccination program increases the protection rate by up to 80% [9, 10] . Different studies indicate that there is no proper protection against this genotype through routine IB vaccination programs. One of the best strategies in controlling IBV is the preparation of autogenous vaccines from the dominant circulating strain and its use in breeders, which will transfer maternal antibodies to offspring and protect them during the production phase. This study aims to develop and provide the autogenous variant 2 vaccine and to determine the immunogenicity of this vaccine. 
The vaccine strain was selected based on molecular epidemiology studies as well as molecular characterization from the Faculty of Veterinary Medicine, University of Tehran.
The virus was isolated from broiler flocks in 2015 and was confirmed by the sequencing of spike gene and full genome characterization [7] . The tenth passage of this virus was selected. 50% of embryo infectious doses (EID50) was calculated using the Spearman-Karber method. [11] .
The harvested allantois fluid was cultivated in aerobic and anaerobic bacterial and fungal growth media and was examined daily for a week in terms of any microbial growth. To determine the purity of the virus, a PCR test was conducted for Avian Influenza, Newcastle Disease, in addition to Mycoplasma spp. , and Avian adenovirus [12] .
The virus was inactivated with 37% formaldehyde (concentration of 0.1%) for 24 h at 37 °C. To confirm the inactivation of the virus, samples from each batch were randomly taken and injected into eggs and passaged at least three times. After complete inactivation, inactivated viruses were emulsified at a ratio of 30 to 70 (w/w) in an aqueous phase from the ISA-70 mineral emulsion (SEPPIC, Cosmetics/Pharmacy Division, France) according to manufacturer's instructions. Each dose of the vaccine contained 0.5 ml (10 6.5 EID50 ) of vaccine emulsion, similar to the commercial vaccine [12] .
One dose of the vaccine was subcutaneously injected into each of the ten 21-days-old white specific pathogen-free (SPF) chickens. These chickens were examined for abnormal reactions for 21 days after injection [12] .
One hundred SPF chickens were divided into 5 groups (Table 1 ) of 20, including the H120+variant 2 vaccine group, the H120+ inactive commercial vaccine (M41) group, the H120 group, and the two control groups. In order to prime the immune system, all groups (except control groups) were vaccinated with live attenuated H120 vaccine on day one.
Then they received an inactivated subcutaneous vaccine three weeks after priming. The control group received 0.5 ml of PBS. Three weeks after vaccination, the chickens were sampled for antibody titer (IDEXX) and challenged with variant 2 viruses (10 3 EID50) oculonasal. On the 5 th day after the challenge, chickens of each group were euthanized.
Their trachea was carefully removed and examined for the activity of the tracheal respiratory cilia. Moreover, the viral load in the kidney and the feces (15 samples/each group) was evaluated using Real-Time quantitative RT-PCR [13] .
Assessment of protection against challenge in each experiment was evaluated using ciliostasis test [14] . Five days post-challenge, all of the chickens in each group were euthanized. The tracheas were carefully removed and examined for ciliary's activity, as described previously [15] . Briefly, each of 10 tracheal rings (3 rings of upper, 4 rings of middle and 3 rings of the lower trachea) prepared from each trachea was examined by low-power microscopy and ciliary activity scored as follows: 0=all cilia beating; 1=75% beating; 2=50% beating; 3=25% beating; and 4=none beating (100% ciliostasis). This gave a maximum possible ciliostasis score for a trachea of 40 in case of complete ciliostasis (total lack of protection).
In this experiment, GraphPad Prism software was used for statistical analysis (One Way ANOVA). The p-value lower than 0.05 was considered significant.
Inactivation of the virus was confirmed. Inoculated embryos showed no specific IBV symptoms such as fatality, dwarfism, stunting, and torsion after at least three passages.
The PCR test results were negative for viruses (AI, ND, and Adenovirus) and Mycoplasma spp. In a stability test after a period, there was no physical change in vaccine suspension, and, with a slight shake, the vaccine regained its former emulsion status. Therefore, the vaccine will be stable for a week at 37° C and for least 3 months at 4°C without any physical changes. The results of the immunization test revealed that these vaccines were safe for chickens, and none of the inoculated chickens showed local or systemic abnormal reactions . The titer results from the ELISA test showed that the ELISA titer derived from the commercial vaccine (M41) was 4882±91.64, and the autogenous vaccine was 4503±160. This difference in antibody titers was significant (p <0.05). The group that received only one H120 vaccine also had a titer of 996.08 ± 38.4 (Figure1).In the Real-Time RT-PCR test of the kidneys, the average CT in the varniat2 group was (33.1±1.5), while in the commercial vaccine group, it was (30.13±1.51), and in H120 vaccine group was (25.86±1.72) (non-significant) ( Figure 2) . Also, the average CT of the Real-Time RT-PCR test on fecal swabs is (28.2 ±1.6) for the group that received the variant2 inactive vaccine and (22.68± 1.82) for commercial vaccine group was (non-significant) ( Figure 3 ).
These results indicate the proper functioning of the autogenous vaccine protection of the kidneys. Although the difference was non-significant (p>0.05), the ciliostasis score in the J o u r n a l P r e -p r o o f autogenous vaccine group was (13 ± 2.2), which was less than that in the commercial vaccine (16.13±2.64) (non-significant) (p> 0.05) (Figure 4 ).
Infectious Bronchitis (IB) is a contagious viral infection that can infect chickens of all ages.
The pathogenicity of this virus is related to chickens' respiratory, reproductive, and urinary system that leads to weight loss, decrease in egg production, and deformation, ultimately leading to increased bird losses, especially in industrial production. As previously described, the virus tends to mutate easily, and its recombination property results in antigenic changes [16] . One of the problems in inactivated vaccines' production is field trial evaluation due to the high costs of testing and the lack of appropriate facilities. The test is time-consuming and costly, depending on which production cycle the birds are in (the nature of the work is in the isolator). There are various types of strains of IB in inactivated vaccines worldwide.
The most famous genotype used is M41. Some commercial vaccines have different J o u r n a l P r e -p r o o f variants in addition to the standard M41 strain, including the Dutch strains (D1466/D274). In an unpublished study conducted by the authors, it was shown that the addition of these Dutch strains could provide a high level of protection (non-significant).
According to the present study, if the strain of variant 2 is added to the vaccine as a strain of the variant with M41 standard strain, the flock will be better protected. The study results indicated that since the autogenous vaccine was produced to prevent respiratory disease in chickens, the vaccine produced from Variants 2 and M41 had better protection against variant 2 in comparison to H120. These results were confirmed by investigating the antibodies titers in chickens' blood sera. Although the protection difference between M41 and variant 2 vaccine was not statistically significant, even a 7% increase in protection rate in the farms could be considered significant. According to the results, the application of the autogenous vaccine can be very useful, especially in combination with the M41 strain. With this combination, we can expect flock protection during the production phase (Layers and breeders) and, more importantly, the protection of chicks to whom the maternal antibodies will be transferred.
No conflict of interest is declared.
The procedure has been conducted according to the University of Tehran 's instructor's guide and ethical standards for treating animals. 