Infectious bursal disease is a highly contagious disease caused by the infectious bursal virus (IBDV) of the avian double RNA virus genus in the family of double RNA viruses. It is acute and accompanied by severe immune suppression, mainly affecting young chickens and causing significant economic impact. The disease first broke out in 1957 and was officially reported in the United States in 1962. The early characteristic of this disease was extensive kidney damage, which was then known as avian kidney disease. The huge economic losses caused by this disease come from two factors. It destroys the developing B lymphocytes containing IgM in the bursa of Fabricius, causing severe immune suppression, reducing humoral immune response ability, making chickens more susceptible to infection, and easily leading to vaccine failure; Three week old or young chickens have a mortality rate of over 60%. The incubation period of this disease is short, about 3-5 days. Chickens less than 3 weeks old have no clinical symptoms, but it leads to immune suppression. The degree of immune suppression caused by IBDV depends on the age of the infected chickens, with 2-week-old chickens experiencing more severe immune suppression than older chickens.

I. Genomic Structure and Pathogenesis

The genome of infectious bursal disease virus is composed of segmented double stranded RNA (dsRNA), which is divided into A and B segments. The virus particles are 55-60nm, with no envelope and a symmetrical icosahedral capsid. The B fragment is relatively small and encodes viral protein 1 (VP1, 95kDa). The A fragment (2.9kb-3.4kb) has two partially overlapping open reading frames (ORFs). Among them, the smaller ORF encodes the non structural protein VP5 (17kDa), which is associated with the pathogenicity of the virus and its transmission between cells; The larger ORF encodes a 110 kDa polyprotein, which self lyses to produce three peptides: pVP2（48kDa）、VP3（32kDa）、VP4（28kDa）。 VP4, as a protein with serine lysine protease activity, mediates this process.

The VP1 protein is an RNA dependent RNA polymerase that exists in a free form in viral particles, and this genome junction protein (VPg) attaches to the 5 'end of the positive strand of two genome fragments. Research has shown that the VP1 gene sequence of the highly virulent vvIBDV pathogen forms a unique cluster, indicating that it may originate from gene recombination of the B segment. The VP1 protein regulates viral virulence due to its role in viral replication efficiency, and virus replication is inhibited through RNA (DNA vector based) in vitro interference experiments targeting the VP1 protein.

Outer shell protein VP2 is the main structural protein, accounting for approximately 51% of the total viral protein (Heetal, 2009). It has at least 2 neutralizing epitopes that can induce the production of neutralizing antibodies. It is responsible for antigen variation, tissue adaptation ability, and viral virulence. The two short peptide products during the maturation process of VP2 determine the assembly and destruction of virus particles on the cytoplasmic membrane during virus attachment and cytoplasmic translocation. Proteins are folded into three key structural domains: substrate, shell, and protrusion. VP2 is not the only virulence determinant in super toxicity, and VP1 protein is another virulence determinant (Qi et al., 2013).

The underwear shell protein VP3 (32kDa) is a Y-shaped trimer, accounting for about 40% of the total viral protein, forming the internal scaffold of the viral protein assembly. It has group specificity and a small number of neutralizing antigenic epitopes, interacts with VP1, and interacts with viral genomic material through its carboxyl terminal domain. Some scholars believe that the VP3 protein promotes virus replication and progeny virus production by interacting with almost all components of the virus particle, including itself, VP2, VP1, and two genomic dsRNAs.

VP4 (28kDa) is a viral autocatalytic protease, which is a soluble protein that utilizes serine lysine catalyzed dimers without ATPase domains to cleave polyprotein into individual VP2, VP3, and VP4 proteins. VP4 is conserved in IBDV serotypes and strains. This protein plays an important role in the maturation of pVP2 protein by continuously modifying several small peptides at the C-terminus during assembly.

The virus non structural protein VP5 is a class II membrane protein with cytoplasmic N-terminal and extracellular C-terminal domains. It has strong alkalinity and semi conservation in all IBDV serotype 1 strains, and is rich in cysteine. VP5 can induce lesions in the bursa of Fabricius and plays an important role in virus transmission and release. The protein accumulates within the cell membrane, leading to a decrease in cell viability. VP2 and VP5 can induce cell apoptosis in vitro culture.

II. Antigen Drift and Genomic RNA Mutation

Outer shell protein VP2 can induce the production of neutralizing antibodies. The region where the antigen epitope is located on VP2 exhibits high nucleotide variability, indicating its susceptibility to antigen conversion and drift. Research has shown that the virulence, tissue tropism, and pathogenic phenotype of highly virulent vvIBDV are determined by certain amino acid residues at positions 253, 279, and 284 in the VP2 protein. Reverse genetics research data shows that a single mutation at position 253 or any other amino acid within the VP2 hypervariable region is sufficient to alter the virulence of IBDV. In vvIBDV isolated from chickens vaccinated with the classic strain vaccine, a glycine serine mutation of G254D was observed, indicating that this mutation may lead to vaccine failure. Epidemiology has discovered a novel and unique strain of IBDV, which has unique AA sequences 272T and 289P in the VP2 hypervariable region and 234P in VP1. These sequences are conserved and, based on molecular characteristics and pathogenicity, are referred to as the novel IBDV (dIBDV). It is widely distributed in South America, Europe, and Asia.

Based on the genetic diversity of IBDV VP2 molecular epidemiology, scholars suggest using a new classification method to divide it into 7 genotypes. Some genotypes have global distribution, such as members of genotype 1 (caIBDV) and genotype 3 (vvIBDV and its recombinant strains), while members of genotype 2 (vaIBDV discovered in the Americas), genotype 4 (dIBDV discovered in South America), and genotype 5 (discovered in Mexico and considered a recombinant strain of caIBDV and vaIBDV) have regional distribution. The genotype 6 comes from the Middle East (Saudi Arabia) and has a 92-93% correlation with the ITA genotype in Italy and a 94-95% correlation with the IBDV RF-5/94 strain in Russia. The members of genotype 7 mainly come from Australia and a few from Russia.

III. Serotype and Pathogenicity

IBDV is divided into serotype 1 and serotype 2 on antigen. Serum type 2 mainly infects turkeys, has no toxicity to chickens, and cannot provide cross protection against serum type 1. Serum type 1 has different pathogenicity to chickens due to differences in virulence, immune suppression, and antigenicity. The early IBDV outbreaks were mainly caused by the classic strain (caIBDV), with inflammation, swelling, and then atrophy of the bursa of Fabricius. In the early 1980s, a variant strain (vaIBDV) emerged in the United States, Central America, and Australia. It differs from classical or super virulent strains in terms of antigen and only causes atrophy of the bursa of Fabricius without inflammation. In the late 1980s, a highly virulent strain (vvIBDV) emerged in Western Europe, Southeast Asia, and Africa, characterized by large cysts and then atrophy. Its virulence was stronger than the classical strain, and the mortality rate exceeded 70%. Classic and virulent strains can cause hemorrhagic inflammation accompanied by severe follicular depletion of the bursa of Fabricius, with only a difference in mortality rate (caIBDV is 30-60%, vvIBDV is 70-100%). The mutant strain causes rapid atrophy of the bursa of Fabricius without inflammation or bleeding, with a mortality rate of less than 10% or none. Chickens vaccinated with the caIBDV vaccine can still be infected with the mutant strain. Mutant and super strong strains, as well as the recent novel IBDV (dIBDV), can break through maternal antibodies and infect young chickens, with a mortality rate of up to 60% in broiler chickens and 25% in broiler chickens. Although the super strain has antigenic similarity with the classical strain, chicken flocks with high maternal antibodies against the classical strain can still be infected with the super strain.

Bleeding of the bursa of Fabricius

Large French cyst

At present, IBD vaccines are mainly inactivated or live vaccines. Chickens that have been vaccinated with inactivated or attenuated live vaccines are protected against IBD through maternal antibodies. However, maternal antibodies can interfere with the effectiveness of attenuated live vaccines, unless moderate and virulent vaccines are used, which can cause damage to the bursa of Fabricius caused by the vaccine and lead to immune suppression. Therefore, there is an urgent need to develop safe and effective vaccines that can induce the correct immune response even in the presence of maternal antibodies.

IV. Innate immune response

Experiments have found that attenuated live vaccines and inactivated vaccines developed using mutant strains have cross protective effects on chickens infected with classical or mutant strains; The vaccine developed using the classic IBDV strain partially protects or cannot protect against variant infections.

Gene expression studies have shown that acute infection with IBDV activates bursa T cells and splenic macrophages. The depletion of bursa cells leads to upregulation of genes associated with NK cell, macrophage, and T cell activation, including interferon, interleukin, and MIP-1, as well as genes regulating the innate immune system such as MD-1 MD-2、 Complement, heat shock proteins, as well as inflammatory and pro-inflammatory response genes.

Genetic regulation of innate immune response during IBDV infection

IBDV can be detected in mononuclear macrophages in the intestine 8-12 hours after oral infection, and these cells then transport the virus to the bursa of Fabricius for effective virus replication in IgM carrying B cells. Infection activates the NF kB pathway and other intracellular signaling pathways. Massive infiltration of macrophages into the bursa of Fabricius can induce high expression of pro-inflammatory mediators such as interleukin. In the study of IBDV, high expression of IFN, chemokines, complement components, and defense factors was observed in central lymphoid organs. Experimental studies have found that NK cells may be involved in the pathogenesis and innate immune response of IBDV infection.

Li Xiaoqing

2023.03.08