Landscape of IGH germline genes of Chiroptera and the pattern of Rhinolophus affinis bat IGH CDR3 repertoire

Abstract

The emergence and re-emergence of abundant viruses from bats that impact human and animal health have resulted in a resurgence of interest in bat immunology. Characterizing the immune receptor repertoire is critical to understanding how bats coexist with viruses in the absence of disease and developing new therapeutics to target viruses in humans and susceptible livestock. In this study, IGH germline genes of Chiroptera including Rhinolophus ferrumequinum, Phyllostomus discolor, and Pipistrellus pipistrellus were annotated, and we profiled the characteristics of Rhinolophus affinis (RA) IGH CDR3 repertoire. The germline genes of Chiroptera are quite different from those of human, mouse, cow, and dog in evolution, but the three bat species have high homology. The CDR3 repertoire of RA is unique in many aspects including CDR3 subclass, V/J genes access and pairing, CDR3 clones, and somatic high-frequency mutation compared with that of human and mouse, which is an important point in understanding the asymptomatic nature of viral infection in bats. This study unveiled a detailed map of bat IGH germline genes on chromosome level and provided the first immune receptor repertoire of bat, which will stimulate new avenues of research that are directly relevant to human health and disease.IMPORTANCEThe intricate relationship between bats and viruses has been a subject of study since the mid-20th century, with more than 100 viruses identified, including those affecting humans. While preliminary investigations have outlined the innate immune responses of bats, the role of adaptive immunity remains unclear. This study presents a pioneering contribution to bat immunology by unveiling, for the first time, a detailed map of bat IGH germline genes at the chromosome level. This breakthrough not only provides a foundation for B cell receptor research in bats but also contributes to primer design and sequencing of the CDR3 repertoire. Additionally, we offer the first comprehensive immune receptor repertoire of bats, serving as a crucial library for future comparative analyses. In summary, this research significantly advances the understanding of bats' immune responses, providing essential resources for further investigations into viral tolerance and potential zoonotic threats.

Keywords: IGH; bat; germline gene; immune repertoire.

Fig 1

Structure of bat IGH loci and the three clan of V genes. (A) The IGH locus of Rhinolophus ferrumequinum. (B) The IGH locus of Phyllostomus discolor. (C) The IGH locus of Pipistrellus pipistrellus. (D) The phylogenetic tree of Pipistrellus pipistrellus. (E) The phylogenetic tree of Phyllostomus discolor. (F) The phylogenetic tree of Rhinolophus ferrumequinum. Green segment is IGHV gene; light blue segment is IGHD gene; yellow segment is IGHJ gene; dark blue segment is IGHC gene.

Fig 2

The structure of IGHJ genes and RSS sequence. (A) Sequence comparison of all IGHJ genes in three bat species. (B) RSS characteristics of V and J genes in bat, human, and mouse. (C) RSS characteristics of D genes in bat, human, and mouse.

Fig 3

Alignment of Rhinolophus affinis sequence with that of annotated three bat species germline genes. (A) Partial sequences of Rhinolophus affinis. (B) The proportion of V, J, and C genes of three annotated bats in the Rhinolophus affinis. (C) The comparison of C region between Rhinolophus ferrumequinum and Rhinolophus affinis. (D) Sequence comparison of Cytb gene in four bats. All the sequences of Rhinolophus affinis were obtained by sequencing.

Fig 4

Analysis of Rhinolophus affinis IGH CDR3 repertoire. (A) V/J gene access of Rhinolophus affinis, human, and mouse. (B–D) V-J pairing in Rhinolophus affinis, humans, and mouse, respectively (only one sample of each species randomly selected for display, and the rest is shown in Supplement figure). (E) CDR3 length distribution of IGH subclasses in Rhinolophus affinis, humans, and mouse, respectively. (F) The insertion and deletion of CDR3 region of Rhinolophus affinis, humans, and mouse, respectively. (G) The CDR3 length composition of Rhinolophus affinis, humans, and mouse, respectively. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

The clones analysis. (A) Distribution of rare clones in Rhinolophus affinis, humans, and mouse, respectively. (B) Statistics of clones above 100 in Rhinolophus affinis, humans, and mouse. (C) The Shannon index of Rhinolophus affinis, humans, and mouse. (D) The shared clones of Rhinolophus affinis, humans, and mouse, separately. (E) The clonotype tracking between Rhinolophus affinis and mouse. B, bat; H, human; M, mouse. ns, P > 0.05; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 6

The AA usage and motif composition of CDR3 region and mutations in IGHJ region. (A) Statistics of AA usage of IGH subclasses in Rhinolophus affinis, humans, and mouse, respectively. (B) The top 10 motifs of IGH in Rhinolophus affinis, humans, and mouse, respectively. (C) Statistics of mutations in IGHJ region of Rhinolophus affinis, humans, and mouse. B, bat; H, human; M, mouse. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.