Table 1.

IBV genotyping and serotyping systems

General classification	Typing method	Test	Description and assessment parameters	Advantages and disadvantages	References
Functional	Immunotyping/ protectotyping	Cross-immunization test	Assesses cross-immunity in chickens following challenge by: 1. Clinical signs 2. Virus re-isolation 3. Histopathological changes 1. Ciliostasis test 2. Detection of viral genome (RT-PCR) 3. Serology	Adv.: 1. Direct information about vaccine efficacy 2. The best protocol to determine cross-protection between IBV strains 3. Whole immune responses are involved in the test Disadv.: 1. Laborious and high cost 2. The vaccine application methods, the challenge virus, assessment parameters, type and age of bird influence the results.	[30, 111, 112]
		Cross-immunization Test	Assesses cross-immunity in TOC by in vitro ciliostasis test	Adv.: 1. Useful for comparing tissue tropism 2. More economical than cross-immunization studies 3. The protocol is better standardized than cross-immunization. 4. Less labor-intensive than cross-immunization tests Disadv.: 1. Complex methodology 2. Requires highly trained technicians 3. Some IBV variants show different behavior in TOC and chickens. 4. The immune system is not involved.	[29, 113]
	Antigenic typing/ serotyping	Virus neutralization test	Assesses the neutralizing reaction between specific antisera against the unknown isolates in eggs, TOC, or cell culture	Adv.: 1. More sensitive test than HI Disadv.: 1. Lack of standardization 2. Time-consuming and laborious 3. VNT is less accurate than cross-immunization (lack of internal controls)	[30, 114]
		Hemagglutination inhibition (HI)	Assesses the neutralizing reaction between specifically known antisera against unknown isolates in using HI test	Adv.: 1. Simple test 2. Less expensive than VNT 3. Less time-consuming than VNT Disadv.: 1. Lack of standardization. 2. Higher cross-reactivity between strains than VNT	[35, 115]
	Antigenic typing (epitope-typing)	Monoclonal antibody	Neutralization of IBV samples using specific monoclonal antibodies in eggs, cell culture or TOC	Adv.: 1. Useful in rapid diagnosis and epidemiological studies 2. Useful in dissecting the virion and elucidating functional relationships Disadv.: 1. Higher risk of false-negative results 2. Needs confirmation by other serotyping tests 3. Sophisticated technique that needs epitope identification and mapping	[37, 116–118]
Non-functional genomic		Genotype-specific RT-PCR	Genotype-specific oligonucleotide primers	Adv.: 1. Accurate and fast technique 2. Cost-effective Disadv.: 1. Unable to differentiate between vaccine and field strains	[119–121]
		PCR + restriction enzyme fragment length polymorphism (RFLP)	The S1 gene PCR product is digested with restriction enzymes. The RFLP patterns are compared with the patterns of reference serotypes.	Adv.: 1. Fast typing method for rapid diagnostics 2. Genotyping can be done very quickly compared to serotyping 3. A large number of samples can be tested Disadv.: 1. Mutations with no relevance for the antigenic or biological function of the virus in cleavage sites may impair results. 2. Correlation with biological and functional properties is uncertain 3. Does not give reliable data about antigenicity 4. Subsequent tests needed for identification of field isolates 5. Mixed strains yield difficult-to-read restriction patterns.	[119, 122, 123]
		RNase T1 fingerprinting	IBV genome digestion with RNase T1 to resolve the resulting oligonucleotide in 2D gel electrophoresis to determine the specific fingerprint of the genome in comparison to reference genotypes	Adv.: 1. It gives information about the whole genome. Disadv.: 1. Results cannot be translated into antigenic or biological function. 2. Different serotypes give distinct fingerprints, but within serotypes, different results might be obtained if the genome identity is less than 95%. 3. Complex technique and labor-intensive	[124, 125]
		Sequencing	Partial or full genome sequencing to compare nucleotide and amino acid sequences and to conduct phylogenetic, recombination, and/or phylodynamic analysis	Adv.: Accurate and fast technique Disadv.: 1. Relatively high cost 2. Sophisticated and expensive software is required. 3. Mutations in the RNA may not correlate with biological or functional changes	[20]
Metagenomic			Structural analysis of the most antigenic protein (S protein) to determine changes in ??receptor-binding domain?? or epitopes	Adv.: 1. Better insight into the effect of changes in RNA sequence on the biological or functional characteristics 2. Reduces the usage of laboratory animals. 3. Useful in the development of structure-based vaccines Disadv.: 1. High cost (equipment, sophisticated software and hardware) 2. Requires epitope mapping	[22, 126, 127]