In the present study, we have clarified the genogroups among the field isolates and produced vaccines and shown the persistent predominance of the A3-genotype in Vietnam since 1987. The long-lasting A3- and in particular, circulating A3(B3)-strains in Vietnam confirmed their genetic adaptibility to cause “very virulent” outbreaks even in the “intermediate plus” vaccinated poultry flocks. The study also demonstrated an important hallmark for the evolutionary distance within and between genotypes and the genotype-typical signature motifs in the strain’s genotypic distinguishability. The existence of the A1a and A1b sub-genotypes and the rare A7-genotype case shed light on the early introduction of these less virulent IBDVs into Vietnam. The preliminary identification of the B1 and B3 genotypes also marked the adequate grouping between the two A and B segments in the Vietnamese IBDV population.
The genotyping system proposed by Jackwood et al. [19], as well as the unified genotypic classification scheme proposed by Islam et al. [12], comprehensively identified the bi-segmented genomic IBDVs into genogroups such that segment A can be divided into A1 (which corresponds to G1, or cIBDV, including A1a, or classically virulent, and A1b, or classically attenuated subgenotypes), A2 (or G2, American variant, varIBDV), A3 (or G3, very virulent, vvIBDV), A4 (or G4, distinct, dIBDV), A5 (or G5, Mexican, MEX-IBDV), A6 (or G6, Italian, ITA-IBDV), A7 (or G7, Early Australian, AUS-earlyIBDV), and A8 (Australian variant, AUS-varIBDV), and A0 for strains of serotype 2, while all IBDV strains of both serotypes 1 and 2 are concisely classified into five B-genotypes of B1 to B5 [12]. This unified genotyping system proved to be practical to evaluate the genetic diversity within and between genotypes and serotypes, and was able to clarify the reassortants and recombinants of the IBDV strains. The unified genotypic classification scheme based on both segments A and B has been effectively applied for the genotyping of the IBDVs in many countries worldwide [12, 17, 20, 22].
We performed the genotype classification and molecular characterization of the 1987–2021 Vietnamese IBDV strains isolated from 18 provinces and some vaccines produced in Vietnam according to the newly unified genotypic scheme by phylogenotyping and sequence analyses. In this study, we have shown the genotypic diversity and clarified three A genogroups, the A1, A3, and A7, and two B genogroups, B1 and B3, that appeared to be present in the field IBDVs throughout the country. The study also revealed continuous molecular genetic evolution and the dominance of A3-genotype strains across the country.
In our study, nine strains isolated from fields were associated with the 2512 Winterfield intermediate plus vaccines (DQ355819-2512VacUS and DQ355820-BlueVacUS) and placed in the A1aB1 group (classical virulent) and two others in the A1aB1 (classical attenuated) group (Fig. 2). These A1a-classical virulent viruses were continually detected in the field isolates (2001–2021), all indicated their origination of the 2512 Winterfield vaccine deravatives which have been used for vaccination since the 1990s [24, 25]. Classically virulent (genogroups A1aB1 and A1aB3) and classically attenuated (genogroup A1bBx) strains were also identified in recent Vietnamese field isolates (2018–2021), indicating co-existence with A3B(1/3) and demonstrating the complicated reassortment between A-genotypes (A1 and A3) and B-genotypes (B1 and B3) in circulating IBDVs in Vietnam.
One of the early isolated A3-genotype strains first reported in Vietnam was the strain G202wt (or G202; GenBank: FJ842491 for segment A) in 1987 in Hanoi (Table 1) [25]. In an investigation by To et al. [24], 19 “very virulent” (A3) and four “classically virulent” (A1) isolates were identified in the different outbreaks in Hanoi and Ho Chi Minh City between 1997 and 1998. All the findings in the present and previous studies have implied the predominance of the A3-genotype strains over the A1-genotype strains from 1987 to 2021. The A3-strains are still persistently dominant in the circulating IBDVs that were detected in fields in the last five years (2016–2021) (Table 5). Not only were the A3-genotype and A3B3 strains (segment A very virulent-like/segment B early Australian-like) of the persistent dominance detected in Vietnam [24, 25], China [3, 17, 22, 23, 34, 37, 38],
Korea [39], Malaysia [26], India [40], Pakistan [41, 42], and Bangladesh [12, 20], but it has also been observed in many European [18, 43], African [44–47], and North/South American countries [16, 48–50]. These A3B3 genotype viruses were apparently emerged from the early reassortment of the "very virulent" segment A of the European strains around the 1980s and 1990s [51, 52] and the "early-Australian" vaccine-originated segment B of the Australian IBDVs around the 1970s [8, 33, 53, 54]. Our 1987-isolated G202wt strain in Vietnam was the most historic isolate of the A3–genotype, which might be introduced from the 1980s-IBDV European source.
The possibility of such reassortment to result in isolates with segment A from the "very virulent" (A3) and segment B derived from the classically attenuated IBDVs (B3) (or vaccines, i.e., from the 002–73 Australian strain) [54] has been well documented, and this co-evolution occurred due to the co-existence of the virulent and attenuated vaccine strains in the field [20, 37, 55–57]. Why the A3B3 reassortants have become so virulent and widely spread worldwide, and how the B3 (the early Australian-like B segment) was reassorted with the A3 segment, is a question for further investigation. However, as genetic evidence from close genotype relationships of viruses between Asian countries—first Vietnam, Korea, China, and Australia—suggests, the possibility of IBDV transboundary transmission via poultry and their products traded in these countries is not ruled out.
The identified A1, A3, and A7 genotypes from the Vietnamese IBDV strains have shown very low evolutionary divergence (2.5–4.2%) within the reference A1, A3, and A7 group mean distances, while between the genotypes the mean distances indicated a high (8.6% for A1 and A3), higher (11.9% for A3-A4 to 12.1% for A1-A6), and the highest divergence (20.5% for A6-A7 to 21.7% for A5-A7) (Tables 2 and 3). The segment B exhibits a higher distance between B-genotypes, ranging from 14% between B1 and B3 to 17.3% between B2 and B4. These mean that the A- and B-genotypes certainly maintain the average intergenotypic nucleotide divergence, which is proposed to be about 9% or higher for A- and 12% or higher for B-segment evolutionary distances [12].
The IBDV VP2 has 1356 nucleotides, encoding 452 amino acids, in which the HVR possesses the key epitopic residues streched within the hydrophilic domains that interact in important ways with the host immune system and responsible for distinghuishable genetic variation between genotypes [8, 11, 25]. Certain signature motifs of residues at positions 222, 253, 256, 294, and 299 in the HVR region have been characterized typical for each genetic groups that distinguish different genotypes. The such motif was early identified as 222A - 253Q - 256I - 294I - 299S for the A3-genotype [8]. Of 52 Vietnamese field A3-genotype strains, that were studied, 50 carried the common A3-genotype typical motifs (222A253Q256I294I299S) and this motif was seen unique in 26 reference A3-genotype strains (Table 4). However, mutations within the signature motif were observed in two of the Vietnamese A3-genotype strains isolated in 2021 (GVP2A-2021-Vietnam and GVP3-2021-Vietnam). In these two strains, the A3-motif changed to (222A253Q256(V/I)294I299N). Eleven Vietnamese A1a-subgenotype strains have a unique motif (222P253Q256V294I299N) that makes an interesting retain of the 294I residue that is present in all the Vietnamese and reference vvIBDV strains.
A comparison of 141 HVR amino acid sequences representing all of the A1-A8 genotypes of serotype 1 revealed key residues at positions 222, 253, 256, 294, and 299 that are unique to each genotype and can be used as a signature motif for genotypic discrimination. The most unique sequence for a typical signature motif is the 222A - 253Q - 256I - 294I - 299S for the A3 genotype that are present in all the global HVR sequences and the majority of Vietnamese strains studied. These residues were suggested to be used as a fingerprint for this A3 (G3, very virulent) genotype at the earliest emergence of vvIBDVs [8] and have remained unchanged since then. Although the numbers of strains studied for each are still limited, the sequences are unique and typical for the genotyic discrimination present in the A2, A5, A6, and A8 genotypes. Notably, the A1a/b classical residue 294L was historically conserved in all of these genotypes (Table 4). However, the amino acid change from Q to H at position 253 marked these two subgroups for distinguishing the classically virulent (genotype A1a) and mildly virulent (attenuated, vaccine/genotype Ab1) IBDVs. Understandably, the mutation Q253H was one of the structural changes from the very virulent (253Q, genotype A3) to the 253H, genotype A1b, that is responsible for the adaptation to cell culture during attenuation [58]. Interestingly, residues at 222, 253, and 256 of the A4 genotype (atypical Italian, ITA-IBDVs) that are 222Q - 253E - 256K, extremely distinct from all the remaining genotypes. The most genetic variation occurred at positions 222 (P/S/T/A/Q) and 256 (V/A/I/K/L), which were all located in major hydrophilic peak A and minor hydrophilic peak 1, and mutations at the site 222 and 256 certainly affected the virus’s replication, virulence and antigenicity [11. 25, 35, 36, 58].